TWI782920B - Novel Anti-PCSK9 Antibody - Google Patents

Abstract

The present invention provides a monoclonal antibody against proprotein convertase subtilisin Kexin type 9 (PCSK9), which can block the binding of PCSK9 to LDL receptor, thereby reducing the level of LDL-C. The antibodies of the present invention provide very effective agents for the treatment of a variety of CVD diseases.

Description

Novel Anti-PCSK9 Antibodies

The present invention relates to novel anti-PCSK9 antibodies.

Cardiovascular disease has always been the number one killer threatening human health and life (World Health Organization (WHO), World Health Organization (2011). Various research reports have shown that lowering low-density lipoprotein cholesterol can effectively reduce the risk of cardiovascular disease At present, statins are mainly used to lower cholesterol. However, many patients cannot tolerate high doses of statins due to side effects or cannot effectively control blood lipids with statins (Baigent, C. et al., Lancet 2000, 376(9753), 1670-1681), there is a strong need for new therapeutic approaches in this field. Proprotein convertase subtilisin Kexin type 9 (PCSK9) was first discovered as convertase-1 regulating neuronal apoptosis. It is mainly synthesized in the small intestine and liver (Seidah NG et al., Proc Natl Acad Sci US A 2003;100:928-33). Its prodomain becomes mature PCSK9 after intracellular self-cleavage, and then secreted by liver cells (McNutt, M.C. et al., J . Biol. Chem. 2007, 20(282), 20799-20803). PCSK9 plays an important role in the regulation of cholesterol metabolism. Researchers in two French families with autosomal dominant hypercholesterolemia A function-enhancing mutant gene of PCSK9 was discovered, thereby discovering its role in the regulation of cholesterol metabolism (Abifadel M et al., Nat Genet 2003;34:154-6).PCSK9 mainly binds to the low-density lipoprotein receptor ( LDL-R) is thus decomposed in the liver to achieve the effect of regulating cholesterol levels. In the absence of PCSK9, the low-density lipoprotein receptors on liver cells transport low-density lipoprotein cholesterol to lysozyme for degradation, will be recycled back to the cell membrane surface. When PCSK9 binds to the low-density lipoprotein receptor, it will hinder the recycling of LDL-R and will aggravate its degradation (Verbeek, R., et al., Eur J Pharmacol 2015; Lo Surdo P et al., EMBO Rep 2011;12:1300-5). Several methods of inhibiting PCSK9 are currently being studied, including preventing the combination of PCSK9 and LDL-R by antibody or peptide binding, and inhibiting PCSK9 by gene silencing The synthesis of PCSK9 is inhibited by small molecule drugs (Michel Farnier, Archives of Cardiovascular Disease, 2014, 107, 58-66). The recently approved monoclonal antibody Alirocum Both ab and evolocumab have shown significant effects in lowering low-density lipoprotein cholesterol in Phase II and Phase III clinical studies. Existing data show that, regardless of whether they have received statin therapy, the reduction of low-density lipoprotein cholesterol levels can reach 70% (Dias, C.S et al., J.Am.Coll.Cardiol. 2012, 60(19), 1888-1898 ; Giugliano, R.P et al., Lancet, 2012, 380(9858), 2007-2017; McKenney, J.M et al., J. Am. Coll. Cardiol. 2012, 59(25), 2344-2353).

The present application provides novel anti-PCSK9 monoclonal antibodies (especially fully human antibodies), polynucleotides encoding them and methods of using them. In one aspect, the application provides an isolated antibody or antigen-binding fragment thereof comprising a heavy chain CDR sequence selected from the group consisting of: SEQ ID NO: 1, 3, 5, 13, 15, 17, 25, 27, 29, 37, 39, 41, 49, 55, 57, 59, 67 and 69. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a light chain CDR sequence selected from the group consisting of: SEQ ID NO: 7, 9, 11, 19, 21, 23, 31, 33, 35, 43, 45, 47, 51, 53, 61, 63, 65 and 71. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region selected from the group consisting of: a) a heavy chain variable region comprising SEQ ID NO: 1, SEQ ID NO: 3 and /or SEQ ID NO: 5; b) heavy chain variable region comprising SEQ ID NO: 13, SEQ ID NO: 15 and/or SEQ ID NO: 17; c) heavy chain variable region comprising SEQ ID NO: 25, SEQ ID NO: 27 and/or SEQ ID NO: 29; d) a heavy chain variable region comprising SEQ ID NO: 37, SEQ ID NO: 93 and/or SEQ ID NO: 41; e) A heavy chain variable region comprising SEQ ID NO: 13, SEQ ID NO: 49 and/or SEQ ID NO: 17; f) a heavy chain variable region comprising SEQ ID NO: 55, SEQ ID NO: 57 and /or SEQ ID NO: 59; and g) a heavy chain variable region comprising SEQ ID NO: 1, SEQ ID NO: 67 and/or SEQ ID NO: 69. In certain embodiments, an antibody or antigen-binding fragment thereof described herein comprises a light chain variable region selected from the group consisting of: a) a light chain variable region comprising SEQ ID NO: 7 , SEQ ID NO: 9 and/or SEQ ID NO: 11; b) a light chain variable region comprising SEQ ID NO: 19, SEQ ID NO: 21 and/or SEQ ID NO: 23; c) the light chain can Variable region, it comprises SEQ ID NO:31, SEQ ID NO:33 and/or SEQ ID NO:35; d) light chain variable region, it comprises SEQ ID NO:43, SEQ ID NO:45 and/or SEQ ID NO: ID NO: 47; e) a light chain variable region comprising SEQ ID NO: 51, SEQ ID NO: 53 and/or SEQ ID NO: 23; f) a light chain variable region comprising SEQ ID NO: 61 , SEQ ID NO: 63 and/or SEQ ID NO: 65; and g) a light chain variable region comprising SEQ ID NO: 7, SEQ ID NO: 9 and/or SEQ ID NO: 71. In certain embodiments, the antibody or antigen-binding fragment thereof described herein comprises: a) a heavy chain variable region comprising SEQ ID NO: 1, SEQ ID NO: 3 and/or SEQ ID NO: 5; And light chain variable region, it comprises SEQ ID NO: 7, SEQ ID NO: 9 and/or SEQ ID NO: 11; B) heavy chain variable region, it comprises SEQ ID NO: 13, SEQ ID NO: 15 and/or SEQ ID NO: 17; and a light chain variable region comprising SEQ ID NO: 19, SEQ ID NO: 21 and/or SEQ ID NO: 23; c) a heavy chain variable region comprising SEQ ID NO: 25, SEQ ID NO: 27 and/or SEQ ID NO: 29; and light chain variable region, which includes SEQ ID NO: 31, SEQ ID NO: 33 and/or SEQ ID NO: 35; d) heavy A chain variable region comprising SEQ ID NO: 37, SEQ ID NO: 39 and/or SEQ ID NO: 41; and a light chain variable region comprising SEQ ID NO: 43, SEQ ID NO: 35 and/or SEQ ID NO: 47; e) a heavy chain variable region comprising SEQ ID NO: 13, SEQ ID NO: 49 and/or SEQ ID NO: 17; and a light chain variable region comprising SEQ ID NO: 51 , SEQ ID NO: 53 and/or SEQ ID NO: 23; f) a heavy chain variable region comprising SEQ ID NO: 55, SEQ ID NO: 57 and/or SEQ ID NO: 59; and a light chain variable Region, it comprises SEQ ID NO: 61, SEQ ID NO: 63 and/or SEQ ID NO: 65; Or g) heavy chain variable region, it comprises SEQ ID NO: 1, SEQ ID NO: 67 and/or SEQ ID NO: ID NO: 69; and a light chain variable region comprising SEQ ID NO: 7, SEQ ID NO: 9 and/or SEQ ID NO: 71. In certain embodiments, an antibody or antigen-binding fragment thereof described herein comprises a heavy chain variable region selected from the group consisting of SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 81, SEQ ID NO: 85, SEQ ID NO: 89, SEQ ID NO: 93 and SEQ ID NO: 97 and at least 80% thereof (such as at least 85%, 88%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99%) sequence identity of homologous sequences. In certain embodiments, an antibody or antigen-binding fragment thereof described herein comprises a light chain variable region selected from the group consisting of SEQ ID NO: 75, SEQ ID NO: 79, SEQ ID NO: 83, SEQ ID NO: 87, SEQ ID NO: 91, SEQ ID NO: 95 and SEQ ID NO: 99 and at least 80% thereof (such as at least 85%, 88%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99%) sequence identity of homologous sequences. In certain embodiments, an antibody or antigen-binding fragment thereof described herein comprises a) a heavy chain variable region comprising SEQ ID NO: 73; and a light chain variable region comprising SEQ ID NO: 75; B) a heavy chain variable region comprising SEQ ID NO: 77; and a light chain variable region comprising SEQ ID NO: 79; c) a heavy chain variable region comprising SEQ ID NO: 81; and a light chain A variable region comprising SEQ ID NO: 83; d) a heavy chain variable region comprising SEQ ID NO: 85; and a light chain variable region comprising SEQ ID NO: 87; e) a heavy chain variable region , which includes SEQ ID NO: 89; and light chain variable region, which includes SEQ ID NO: 91; f) heavy chain variable region, which includes SEQ ID NO: 93; and light chain variable region, which includes SEQ ID NO: 93; ID NO: 95; g) heavy chain variable region comprising SEQ ID NO: 97; and light chain variable region comprising SEQ ID NO: 99; or h) with a), b), c), d ), e), f) or g) have at least 80% (such as at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity of heavy chain variable region and light chain variable region. In certain embodiments, the antibody or antigen-binding fragment thereof described herein can be present at no more than 10 ⁻⁷ M, no more than 10 ⁻⁸ M, no more than 10 ⁻⁹ M, no more than 10 ⁻¹⁰ M, no more than A KD value of 10 ⁻¹¹ M or not exceeding 10 ⁻¹² M specifically binds to human PCSK9, and the Kd value is determined by a surface plasmon resonance (SPR) binding method. In certain embodiments, the antibody or antigen-binding fragment thereof described herein can be present at no more than 10 ⁻⁷ M, no more than 10 ⁻⁸ M, no more than 10 ⁻⁹ M, no more than 10 ⁻¹⁰ M, no more than A KD value of 10 ^-11 M or not more than 10 ^-12 M specifically binds to human PCSK9, and the Kd value is determined by ELISA method. In certain embodiments, the antibody or antigen-binding fragment thereof described herein can be present at no more than 10 ⁻⁷ M, no more than 10 ⁻⁸ M, no more than 10 ⁻⁹ M, no more than 10 ⁻¹⁰ M, no more than A KD value of 10 ^-11 M or not more than 10 ^-12 M specifically binds to monkey PCSK9. In certain embodiments, an antibody or antigen-binding fragment thereof described herein can be administered at no more than 2.1 nM (e.g., no more than 3 nM, 2.5 nM, 1.8 nM, 1.7 nM, 1.6 nM, 1.5 nM, 1.4 nM, 1.3 nM IC50 values of nM, 1.2 nM or 1 nM) block the binding of human PCSK9 to its ligand. In certain embodiments, an antibody or antigen-binding fragment thereof described herein can be administered at no more than 0.15 nM (e.g., no more than 0.14, 0.13, 0.12, 0.11, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03 or 0.02 nM) with an EC50 value for binding to human PCSK9. In certain embodiments, an antibody or antigen-binding fragment thereof described herein can be administered at a concentration of no more than 115 nM, no more than 106 nM, no more than 80 nM, no more than 77 nM, no more than 66 nM, or no more than 40 nM. IC50 values (e.g. not exceeding 120 nM, 110 nM, 100 nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 65 nM, 60 nM, 55 nM, 50 nM, 45 nM, 40 nM, 35 nM , 30 nM, 25 nM, 20 nM, 15 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM or 1 nM) restore cellular LDL uptake . In certain embodiments, an antibody or antigen-binding fragment thereof described herein is stable in serum for at least 1 day, at least 3 days, at least 4 days, at least 5 days, at least one week, at least 2 weeks, at least one month . In certain embodiments, an antibody or antigen-binding fragment thereof described herein does not mediate ADCC or CDC or both. In certain embodiments, the antibodies or antigen-binding fragments thereof described herein are fully human monoclonal antibodies. In certain embodiments, the fully human monoclonal antibody is produced by host cells or transgenic animals. In certain embodiments, the antibodies or antigen-binding fragments thereof described herein are capable of reducing LDL cholesterol levels in animals by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% , 50%, 55%, 60%, 65%, 70%, 75%, 77%, 80%, 84%, 85%, 90%, 95% or more. In certain embodiments, an antibody or antigen-binding fragment thereof described herein is capable of maintaining HDL cholesterol levels in an animal. In certain embodiments, an antibody or antigen-binding fragment thereof described herein has a serum half-life of at least 165 hours, at least 250 hours, at least 360 hours, at least 390 hours, or at least 450 hours (e.g., at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 180, at least 200, at least 300, at least 350, at least 400, or at least 500 hours). In one aspect, the application provides an antibody or antigen-binding fragment thereof that competes for the same epitope as an antibody or antigen-binding fragment thereof described herein. In certain embodiments, the present application provides an antibody or an antigen-binding fragment thereof that is a camelized single chain domain antibody, diabody, scFv, scFv dimer, BsFv, dsFv, ( dsFv)2, dsFv-dsFv', Fv fragment, Fab, Fab', F(ab')2, ds diabody, nanobody, domain antibody or bivalent domain antibody. In certain embodiments, the present application provides an antibody or antigen-binding fragment thereof further comprising an immunoglobulin constant region. In certain embodiments, the application provides an antibody or antigen-binding fragment thereof further comprising a conjugate. In certain embodiments, the conjugate can be a detectable label, a pharmacokinetic modifying moiety, or a purified moiety. In one aspect, the application also provides an isolated polynucleotide encoding an antibody or antigen-binding fragment thereof as described herein. In certain embodiments, the polynucleotides provided herein encode the amino acid sequences of the antibodies or antigen-binding fragments thereof described herein. In certain embodiments, the application provides vectors comprising such polynucleotides. In certain embodiments, the present application provides methods for expressing one or more antibodies or antigen-binding fragments described herein by culturing a host under conditions in which an antibody or antigen-binding fragment encoded by a polynucleotide is expressed in a vector cell fulfillment. In certain embodiments, the polynucleotides provided herein are operably linked to a promoter, such as the SV40 promoter, in a vector. In certain embodiments, the host cells comprising the vectors provided herein are Chinese hamster ovary cells, or 293 cells. In one aspect, the present application provides a method of expressing an antibody or antigen-binding fragment thereof as described herein, comprising culturing said host cell under conditions for expressing said polynucleotide. In one aspect, the application provides a kit comprising an antibody or antigen-binding fragment thereof described herein. In one aspect, the application provides a method of treating a disease or condition associated with PCSK9 in an individual comprising administering to the individual a therapeutically effective amount of an antibody or antigen-binding fragment thereof described herein. In certain embodiments, the individual is identified as having a disorder or condition that is likely to be responsive to a PCSK9 inhibitor. In certain embodiments, the individual is identified as having elevated levels of serum LDL cholesterol (LDL-C), total cholesterol, and/or non-HDL cholesterol or decreased levels of LDL receptors in the test biological sample from the individual. In certain embodiments, said LDL-C and/or total cholesterol levels are reduced following administration of said antibody or antigen-binding fragment thereof. In one aspect, the present application also provides a pharmaceutical composition, comprising the antibody or antigen-binding fragment thereof described in the present application and one or more pharmaceutically acceptable carriers. In certain embodiments, the pharmaceutical carrier can be, for example, a diluent, an antioxidant, an adjuvant, an excipient, or a non-toxic auxiliary substance. In one aspect, the application also provides a method of treating a condition in an individual that would benefit from an upregulated immune response comprising administering to the individual an effective amount of an antibody or antigen-binding fragment thereof described herein. In certain embodiments, the individual has upregulated levels of LDL-C, total cholesterol and/or non-HDL cholesterol or downregulated levels of LDL receptors. In one aspect, there is provided a use of an antibody described herein, or an antigen-binding fragment thereof, in the manufacture of a medicament for the treatment of a condition that would benefit from an upregulated immune response. In certain embodiments, the condition is cardiovascular disease, inflammatory disease, and infectious disease. In one aspect, the infectious disease is sepsis.

11.4-VH: 胺基酸序列 (SEQ ID NO: 73):

核酸序列 (SEQ ID NO: 74)

11.4-VL: 胺基酸序列 (SEQ ID NO: 75):

核酸序列 (SEQ ID NO: 76)

18.156.8-VH 胺基酸序列 (SEQ ID NO: 77):

核酸序列 (SEQ ID NO: 78)

18.156.8-VL 胺基酸序列 (SEQ ID NO: 79):

核酸序列 (SEQ ID NO: 80)

15.14.2-VH 胺基酸序列 (SEQ ID NO: 81):

核酸序列 (SEQ ID NO: 82)

15.14.2-VL 胺基酸序列 (SEQ ID NO: 83):

核酸序列 (SEQ ID NO: 84)

17.72.3-VH 胺基酸序列 (SEQ ID NO: 85):

核酸序列 (SEQ ID NO: 86)

17.72.3-VL 胺基酸序列 (SEQ ID NO: 87):

核酸序列 (SEQ ID NO: 88)

18.136.7-VH 胺基酸序列 (SEQ ID NO: 89):

核酸序列 (SEQ ID NO: 90)

18.136.7-VL 胺基酸序列 (SEQ ID NO: 91):

核酸序列 (SEQ ID NO: 92)

19.3.8-VH 胺基酸序列 (SEQ ID NO: 93):

核酸序列 (SEQ ID NO: 94)

19.3.8-VL 胺基酸序列 (SEQ ID NO: 95):

核酸序列 (SEQ ID NO: 96)

40409-VH 胺基酸序列 (SEQ ID NO: 97):

核酸序列 (SEQ ID NO: 98)

40409-VL 胺基酸序列 (SEQ ID NO: 99):

核酸序列 (SEQ ID NO: 100)

在一些實施例中，本申請所述之一或多個CDR序列可經修飾或改變以使獲得的抗體在一或多個性質上相對原來的抗體有所改進(例如改進的抗原結合、改進的糖基化模式、降低的CDR殘基上的糖基化風險、增加的藥代動力學半衰期、pH敏感性及對綴合的相容性)，或與原來的抗體相當(即除上述修飾及改變外具有相同CDR序列的抗體)，或至少實質上保留了原來的抗體的抗原結合特性。 熟習此項技術者應理解，可將表1中提供的CDR序列進行修飾以包含一個或更多胺基酸的取代，由此得到提高的生物學活性例如提高的與人PCSK9的結合親和性。例如，可利用噬菌體展示技術生產且表現抗體變體庫(例如Fab或FcFv變體)，隨後篩選與人PCSK9有親和性的抗體。在另一實例中，可用電腦軟體模擬所述抗體與人PCSK9的結合且鑑別抗體上形成結合界面的胺基酸殘基。可避免此等殘基的替代以防止結合親和性降低，或可靶向此等殘基進行替代以形成更強的結合。在某些實施例中，CDR序列中之至少一個(或全部)取代為保守替代。 在某些實施例中，所述抗體及抗原結合片段包括一或多個CDR序列，此等序列具有與表1中所列的序列至少80%(例如至少85%、88%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%)的序列同一性，且同時保留了與其親本抗體相似或甚至高於其之與人PCSK9的結合親和性，所述親本抗體具有基本相同的序列，但其相應的CDR序列與表1所列的序列具有100%序列同一性。 在某些實施例中，所述抗-PCSK9抗體及其抗原結合片段為全人源的。此等全人源抗體保留了與人PCSK9的結合親和性，較佳與例示性抗體：11.4、18.156.8、15.14.2、17.72.3、18.136.7、19.3.8及40409的水準相似。 本申請亦包括與本申請抗-PCSK9抗體及其抗原結合片段競爭相同抗原決定區的抗體及其抗原結合片段。在某些實施例中，所述抗體以低於10^-6 M、低於10^-7 M、低於10^-7.5 M、低於10^-8 M、低於10^-8.5 M或低於 10^-9 M或低於10^-10 M的IC₅₀ 值(即半數抑制濃度)阻斷11.4、18.156.8、15.14.2、17.72.3、18.136.7、19.3.8及40409與人或猴PCSK9的結合。IC₅₀ 值藉由競爭性測試，例如ELISA測定及放射性配位體競爭結合測定法。 在一些實施例中，本申請所述抗-PCSK9抗體及其抗原結合片段能夠以不超過10^-8 M、不超過10^-9 M或不超過10^-10 M(例如≤2.5×10^-8 M、≤2×10^-8 M、≤7.5×10^-9 M、≤3.5×10^-9 M、≤7×10^-10 M、≤6×10^-10 M、≤5×10^-10 M、≤2.5×10^-10 M、≤2×10^-10 M、≤1.5×10^-10 M、≤7.5×10^-11 M、≤6.5×10^-11 M或≤5.5×10^-11 M)的結合親和性(Kd)與人PCSK9及/或猴PCSK9特異性結合，其藉由表面電漿子共振結合法或ELISA量測。結合親和性可用K_D 值表示，其藉由當抗原及抗原結合分子的結合達到平衡時的解離速率與結合速率的比值(koff/kon)計算得到。所述抗原結合親和性(例如K_D )可藉由此項技術已知的適宜方法適宜地確定，所述方法包括使用儀器如如Biacore的表面電漿子共振結合法(參加例如Murphy, M.等人, Current protocols in protein science, 第19章, 第19.14單元, 2006)。 在某些實施例中，本申請所述抗體及其抗原結合片段與人PCSK9以0.01nM-0.2 nM (例如0.02nM-0.2nM、0.02nM-0.15nM、0.02nM-0.05nM、0.01nM-0.05nM或0.02nM-0.3nM)的EC50 (即半數結合濃度)結合。所述抗體與人PCSK9的結合可藉由此項技術已知之方法，如夾心法，如ELISA，Western印跡或其他結合試驗測定。在例示性實例中，將待測抗體(即一抗)與固定化的人PCSK9結合，隨後洗掉未結合抗體，引入標記的二抗，其能夠與一抗結合因此能夠偵測出結合的一抗。當使用固定化的PCSK9時可在酶標儀板上進行所述偵測。 在某些實施例中，本申請所述抗體及其抗原結合片段以0.5nM-3nM (例如0.5nM-2.5nM、1nM-2.5nM、1nM-2nM或1nM-1.5nM)的IC₅₀ 抑制人PCSK9與其配位體的結合，其藉由競爭性測試測得。 在某些實施例中，所述抗體及其抗原結合片段與猴PCSK9以與人PCSK9相似的結合親和性結合。例如，例示性抗體11.4、18.156.8、15.14.2、17.72.3、18.136.7、19.3.8及40409與猴PCSK9以與人PCSK9相似的親和性或EC₅₀ 值結合。 在一些實施例中，所述之抗-PCSK9抗體及其抗原結合片段亦包括免疫球蛋白恆定區。在一些實施例中，免疫球蛋白恆定區包括重鏈及/或輕鏈恆定區。所述重鏈恆定區包括CH1、CH1-CH2或CH1-CH3區。在一些實施例中，所述恆定區亦可包括一或多個修飾以得到需要的性質。例如，所述恆定區可經修飾以減少或耗竭一或多個效應功能，以增強FcRn受體結合，或引入一或多個半胱胺酸殘基。在一些實施例中，所述之抗-PCSK9抗體及其抗原結合片段具有IgG4同種型的恆定區，其具有降低的或耗竭的效應功能。已知有許多測試用來評估ADCC或CDC活性，例如Fc受體結合試驗、補體C1q結合實驗及細胞裂解法，熟習此項技術者能夠容易選擇。 在一些實施例中，所述抗體及其抗原結合片段可用作抗體-藥物綴合物、雙特異性或多價抗體的基礎分子。 本申請所述之抗-PCSK9抗體及其抗原結合片段可為單株抗體、多株抗體、全人源抗體、全人源抗體、人源化抗體、嵌合抗體、重組抗體、雙特異性抗體、標記抗體、二價抗體或抗獨特型抗體。重組抗體為在體外使用重組方法而非動物製備的抗體。雙特異性抗體或雙價抗體為具有兩種不同的單株抗體的片段的人工抗體，其能結合兩種不同的抗原。「二價」的抗體及其抗原結合片段包括兩個抗原結合位點。兩個抗原結合位點可結合相同抗原，或可各自結合至不同抗原，在此情況下，抗體或抗原結合片段為「雙特異性」。 在一些實施例中，本申請所述之抗-PCSK9抗體及其抗原結合片段為全人源抗體。在一些實施例中，使用重組方法製備所述全人源抗體。例如，可製備轉殖基因動物，如小鼠，使其攜帶人源免疫球蛋白基因的轉殖基因或轉染色體，且因此在用適宜的抗原如人源PCSK9免疫後能夠生產全人源抗體。全人源抗體可自此類轉殖基因動物中分離，或另選地，可藉由雜交瘤技術製備，將所述轉殖基因動物的脾細胞與永生細胞系融合以生成分泌所述全人源抗體的雜交瘤細胞。例示性的轉殖基因動物包括但不限於，Omni大鼠，其內源性大鼠免疫球蛋白基因之表現經失活且同時經基因工程化以包含功能性的重組人源免疫球蛋白基因座；Omni小鼠，其內源性小鼠免疫球蛋白基因的表現經失活且同時經基因工程化以包含具有J-基因座缺失及C-κ突變的重組人源免疫球蛋白基因座。OmniFilc，其為轉殖基因大鼠，其內源性大鼠免疫球蛋白基因的表現經失活，且同時經基因工程化以包含具有單個的共有的、重組的VkJk輕鏈及功能性重鏈的重組人源免疫球蛋白基因座。具體資訊請進一步參見：Osborn M.等人, Journal of Immunology, 2013, 190: 1481-90; Ma B.等人, Journal of Immunological Methods 400-401 (2013) 78-86; Geurts A.等人, Science, 2009, 325:433; 美國專利8,907,157；歐洲專利2152880B1；歐洲專利2336329B1，其均以全文引用的方式併入本申請。亦可使用其他適宜的轉殖基因動物，例如，HuMab小鼠 (具體參見Lonberg, N.等人 Nature 368(6474): 856 859 (1994)), Xeno-小鼠(Mendez等人 Nat Genet., 1997, 15:146-156), TransChromo小鼠(Ishida等人 Cloning Stem Cells, 2002, 4:91-102)及VelocImmune小鼠(Murphy等人 Proc Natl Acad Sci USA, 2014, 111:5153-5158), Kymouse轉殖基因小鼠(Lee等人 Nat Biotechnol, 2014, 32:356-363)，及轉殖基因兔(Flisikowska等人 PLoS One, 2011, 6:e21045)。 在一些實施例中，本申請所述之抗-PCSK9抗體及其抗原結合片段為駱駝化單域抗體(camelized single chain domain antibody)、雙功能抗體(diabody)、scFv、scFv二聚體、BsFv、dsFv、(dsFv)2、dsFv-dsFv'、Fv片段、Fab、Fab'、F(ab')2、ds雙功能抗體(ds diabody)、奈米抗體、域抗體或雙價域抗體。 在某些實施例中，所述抗-PCSK9 抗體及其抗原結合片段進一步包含綴合物。可設想，本發明中的抗體或其抗原結合片段可與多種綴合物連接(見例如「Conjugate Vaccines」、Contributions to Microbiology and Immunology、J. M. Cruse and R. E. Lewis、Jr. (eds.)、Carger Press、New York、(1989))。此等綴合物可藉由共價結合、親和結合、嵌入、同等結合(coordinate binding)、錯合、結合、混合或加入等其他方式與所述抗體或抗原結合物連接。在某些實施例中，本發明揭示之抗體及抗原結合片段可藉由工程化方法使其含有抗原決定區結合部分以外的特定位點，此等位點可用來結合一或多種綴合物。例如，此類位點可包含一或多種反應性胺基酸殘基，例如半胱胺酸殘基及組胺酸殘基，用於協助與結合物的共價連接。在某些實施例中，抗體可間接連於綴合物，或藉由另一個綴合物相連。例如，所述抗體或其抗原結合片段可結合生物素，然後間接結合第二個綴合物，其與親和素相連。所述綴合物可為可偵測之標記、藥代動力學修飾部分、純化部分或細胞毒性部分。可偵測的標記的例子可包括螢光標記(例如螢光素、羅丹明、丹醯、藻紅蛋白或德克薩斯紅)、酶-受質標記物(例如辣根過氧化物酶、鹼性磷酸酶、螢光素酶、葡糖澱粉酶、溶菌酶、糖氧化酶或β-D-半乳糖苷酶)、放射性同位素(例如、¹²³ I、¹²⁴ I、¹²⁵ I、¹³¹ I、³⁵ S、³ H、¹¹¹ In、¹¹² In、¹⁴ C、⁶⁴ Cu、⁶⁷ Cu、⁸⁶ Y、⁸⁸ Y、⁹⁰ Y、¹⁷⁷ Lu、²¹¹ At、¹⁸⁶ Re、¹⁸⁸ Re、¹⁵³ Sm、²¹² Bi、及³² P、其他鑭系元素、發光標記)、發色團部分、地高辛、生物素/親和素、DNA分子或金以進行偵測。在某些實施例中，所述綴合物可為藥代動力學修飾部分如PEG，其幫助延長抗體的半衰期。其他適宜的聚合物包括例如羧甲基纖維素、葡聚糖、聚乙烯醇、聚乙烯吡咯啶酮、乙二醇/丙二醇共聚物等。在某些實施例中，所述綴合物可為純化部分，例如磁珠。「細胞毒性部分」可為對細胞有害的或可能損壞或殺死細胞的任何試劑。細胞毒性部分的例示包括，但不限於，紫杉醇、細胞鬆弛素B、短桿菌肽D、溴化乙錠、吐根鹼、絲裂黴素、依託泊苷、替尼泊苷、長春新鹼、長春鹼、秋水仙鹼、阿黴素、柔紅黴素、二羥基炭疽菌素二酮、米托蒽醌、光神黴素、放線菌素D、1-去氫睾酮、糖皮質激素、普魯卡因、丁卡因、利多卡因、普萘洛爾、嘌呤黴素及其類似物、抗代謝物(例如，甲胺蝶呤、6-巰基嘌呤、6-硫鳥嘌呤、阿糖胞苷、5-氟尿嘧啶達卡巴)、烷化劑(例如氮芥、塞替派苯丁酸氮芥、美法侖、卡莫司汀(BSNU)及洛莫司汀(CCNU)、環磷醯胺、白消安、二溴甘露醇、鏈脲黴素、絲裂黴素C及順-二氯二胺鉑(II)(DDP)順鉑)、蒽環類抗生素(例如柔紅黴素(以前的道諾黴素)及阿黴素)、抗生素(例如更生黴素(以前稱為放線菌素)、博來黴素、光神黴素及氨茴黴素(AMC))以及抗有絲分裂劑(例如長春新鹼及長春鹼)。多核苷酸及重組方法 本申請提供編碼抗-PCSK9抗體及其抗原結合片段的分離的多核苷酸。在某些實施例中，所述分離的多核苷酸包括一或多個如表1中的核苷酸序列，其編碼如表1中的CDR序列。 在一些實施例中，所述分離的多核苷酸編碼重鏈可變區且包括選自下組之序列：SEQ ID NO: 26、SEQ ID NO: 30、SEQ ID NO: 34，以及與其具有至少80%(例如至少85%、88%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%)的序列同一性之同源序列。在一些實施例中，所述分離的多核苷酸編碼輕鏈可變區且包括選自下組之序列：SEQ ID NO: 28、SEQ ID NO: 32、SEQ ID NO: 36，以及與之具有至少80%(例如至少85%、88%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%)的序列同一性之同源序列。在某些實施例中，所述一致性的百分比是源自遺傳密碼的簡併性，而編碼的蛋白序列保持不變。 使用此項技術公知的重組技術，可將包括編碼所述抗-PCSK9抗體及其抗原結合片段(例如包括表1所示的序列)的多核苷酸的載體引入宿主細胞用於選殖(擴增DNA)或基因表現。在另一實施例中，所述抗體可藉由此項技術公知的同源重組之方法製得。編碼所述單株抗體的DNA可藉由習知方法分離及測序(如可使用寡核苷酸探針，該探針可特異性與編碼所述抗體的重鏈及輕鏈的基因結合)。多種載體可供選擇。載體組分通常包括，但不限於，以下的一或多種：信號序列、複製起始點、一或多種標記基因、增強序列、啟動子(例如：SV40、CMV、EF-1α)及轉錄終止序列。 在一些實施例中，所述載體系統包括哺乳動物、細菌、酵母系統等，且將包括質粒例如但不限於pALTER、pBAD、pcDNA、pCal、pL、pET、pGEMEX、pGEX、pCI、pCMV、pEGFP、pEGFT、pSV2、pFUSE、pVITRO,pVIVO、pMAL、pMONO、pSELECT、pUNO、pDUO、Psg5L、pBABE、pWPXL、pBI、p15TV-L、pPro18、pTD、pRS420、pLexA、pACT2等其他可自實驗室獲得或市售的載體。適宜的載體可包括質粒或病毒載體(例如，複製缺陷型逆轉錄病毒、腺病毒及腺相關病毒)。 可將包括編碼所述抗體及其抗原結合片段的多核苷酸的載體引入宿主細胞用於選殖或基因表現。本發明中適用於選殖或表現所述載體中的DNA的宿主細胞為原核細胞、酵母或上述高級真核細胞。適用於本發明用途的原核細胞包括真細菌如，革蘭氏陰性菌或革蘭氏陽性菌，例如，腸桿菌科，如大腸桿菌，腸桿菌屬，歐文氏菌屬，克雷白氏桿菌屬，變形桿菌屬，沙門氏菌屬，如鼠傷寒沙門(氏)桿菌，沙雷氏菌屬，如黏質沙雷氏菌，以及志賀氏菌屬，及桿菌屬，如枯草芽孢桿菌及地衣芽孢桿菌，假單胞菌，如綠膿桿菌及鏈黴菌。 除了原核細胞以外，真核微生物如絲狀真菌或酵母亦可作宿主細胞選殖或表現編碼抗PCSK9抗體的載體。釀酒酵母，或麵包酵母為最常用的低等真核宿主微生物。但是，許多其他屬、種及株均比較常用且在本發明中適用，如粟酒裂殖酵母；克魯維酵母屬宿主如，乳酸克魯維酵母、脆壁克魯維酵母(ATCC 12,424)、保加利亞克魯維酵母(ATCC 16,045)、魏氏克魯維酵母(ATCC 24,178)、克魯雄酵母(ATCC 56,500)、果蠅克魯維酵母(ATCC 36,906)、耐熱克魯維酵母及馬克斯克魯維酵母；解脂耶氏酵母(EP 402,226)；巴斯德畢赤酵母(EP 183,070)；假絲酵母；里氏木黴(EP 244,234)；鏈孢黴；西方許旺酵母，如：西方許旺酵母；及絲狀真菌，如：脈孢菌、青黴菌、彎頸黴及曲黴菌，如：鉤巢麯黴及黑麯黴。 本發明中提供的適用於表現糖基化抗體或其抗原結合片段的宿主細胞由多細胞生物衍生得到。無脊椎細胞的實例包括植物及昆蟲細胞。已發現多種桿狀病毒株(baculoviral strains)及其變體以及對應的許可性昆蟲宿主細胞(permissive insect host cells)，來自於諸如以下的宿主：草地夜蛾(毛蟲)、埃及斑蚊(蚊子)、白紋伊蚊(蚊子)、黑腹果蠅(果蠅)及家蠶。多種用於轉染的病毒株為公眾可得，例如苜蓿銀紋夜蛾核型多角體病毒及家蠶核型多角體病毒的Bm-5變種，此等病毒均可在本發明中使用，特別是用於轉染草地夜蛾細胞。棉花、玉米、土豆、大豆、矮牽牛花、西紅柿及菸草的植物細胞培養亦可用作宿主。 但是，最感興趣的為脊椎細胞，且脊椎細胞的培養(組織培養)已成為習知操作。可用的哺乳動物宿主細胞實例有，SV40轉化的猴腎細胞CV1系(COS-7, ATCC CRL 1651)；人胚胎腎細胞系(293 或懸浮培養的293細胞次選殖，Graham等人, J. Gen Virol. 36:59 (1977))；幼地鼠腎細胞(BHK，ATCC CCL 10)；中國倉鼠卵巢細胞/-DHFR (CHO，Urlaub等人, Proc. Natl. Acad. Sci. USA 77:4216 (1980))；小鼠睾丸支持細胞(TM4，Mather, Biol. Reprod. 23:243-251 (1980))；猴腎細胞(CV1 ATCC CCL 70)；非洲綠猴腎細胞(VERO-76，ATCC CRL-1587)；人宮頸癌細胞(HELA，ATCC CCL 2)；犬腎細胞(MDCK，ATCC CCL 34)；布法羅大鼠肝細胞(BRL 3A，ATCC CRL 1442)；人肺細胞(W138，ATCC CCL 75)；人肝細胞(Hep G2，HB 8065)；小鼠乳腺瘤(MMT 060562，ATCC CCL51)；TRI細胞(Mather等人，Annals N.Y. Acad. Sci. 383:44-68 (1982))；MRC 5細胞；FS4細胞；及人肝癌細胞系(Hep G2)。在某些較佳的實施例中，所述宿主細胞為293F細胞。 用上述的可產生抗PCSK9抗體的表現或選殖載體轉化宿主細胞，且將其在習知營養培養基中培養，所述營養培養基經修飾後適宜於誘導啟動子、選擇轉化細胞或擴增編碼目的序列的基因。 本發明中用於產生所述抗體或其抗原結合片段的宿主細胞可在多種培養基中培養。市售的培養基如Ham's F10 (Sigma)、最低基本培液 (MEM， (Sigma))、RPMI-1640 (Sigma)及Dulbecco's Modified Eagle's Medium (DMEM)，Sigma)可用於培養所述宿主細胞。另外，任何在Ham等人, Meth. Enz. 58:44 (1979), Barnes等人, Anal. Biochem. 102:255 (1980), 美國專利號4,767,704; 4,657,866; 4,927,762; 4,560,655; 或5,122,469; WO 90/03430; WO 87/00195; 或美國專利申請Re. 30,985中說明的培養基均可用作所述宿主細胞的培養基。此等培養基均可添加必要的激素及/或其他生長因子(如胰島素、轉鐵蛋白或表皮生長因子)、鹽類(如氯化鈉、氯化鈣、氯化鎂及磷酸鹽)、緩衝液(如HEPES)、核苷酸(如腺苷酸及胸腺嘧啶)、抗生素(如慶大黴素)、微量元素(定義為終濃度通常在微莫耳範圍無機化合物)，及葡萄糖或與之等同的能量源。所述培養基亦可含有此項技術公知的適當濃度的任何其他必要的添加劑。所述培養基的條件，如溫度、pH值等類似條件，為選擇用於表現的宿主細胞此前所使用的條件，為熟習此項技術者所熟知。 在使用重組技術時，所述抗體可在胞內、壁膜空間生成，或直接分泌至培養基中。若所述抗體在胞內生成，則首先除去宿主細胞或裂解片斷的顆粒殘骸，例如，可藉由離心或超音方法。Carter等人, Bio/Technology 10:163-167 (1992)描述將分泌至大腸桿菌壁膜空間的抗體分離之方法。簡言之，在醋酸鈉(pH 3.5)、EDTA及苯甲磺醯氟(PMSF)存在的條件下化開細胞糊(cell paste)約30分鐘以上。離心除去細胞碎片。如所述抗體分泌至培養基中，則通常首先使用市售的蛋白濃度過濾器，如Amicon或Millipore Pellicon ultrafiltration unit，濃縮該表現系統的上清液。在任何前述的步驟中均可加入蛋白酶抑制劑如PMSF以抑制蛋白降解，以及抗生素以防止偶然污染物的生長。 自所述細胞中製得的抗體可採用純化方法進行純化，例如羥磷灰石層析、凝膠電泳、透析、DEAE-纖維素離子交換層析柱、硫酸銨沈澱、鹽析以及親和層析，其中親合層析為較佳的純化技術。所述抗體的種類以及所述抗體中存在任何免疫球蛋白的Fc結構域決定了蛋白A作為親和配位體是否適合。蛋白A可用於純化基於人γ1，γ2或γ4重鏈的抗體(Lindmark等人, J. Immunol. Meth. 62:1-13 (1983))。蛋白G適用於所有鼠源異構體及人γ3 (Guss等人, EMBO J. 5:1567 1575 (1986))。瓊脂糖為最常用的親和配位體附著基質，但亦可選用其他基質。機械力穩定的基質如可控孔度玻璃或聚(苯乙烯)苯與用瓊脂糖相比可實現更快的流速及更短的處理時間。如該抗體含有CH3結構域，則可用Bakerbond ABX.TM樹脂進行純化(J. T. Baker, Phillipsburg, N.J.)。亦可根據需要獲得的抗體確定其他蛋白純化的技術，如離子交換管柱中的分餾、乙醇沈澱、反相HPLC、矽膠層析、基於陰離子或陽離子交換樹脂的肝素瓊脂糖凝膠層析(如聚天冬胺酸管柱)、層析聚焦、SDS-PAGE、以及硫酸銨沈澱。 在任意初步純化步驟之後，可用低pH疏水相互作用層析之方法處理含有感興趣的抗體及雜質的混合物，用pH約2.5-4.5的洗滌緩衝液，較佳在低鹽濃度下進行(例如，約0至0.25M鹽濃度)。套組 本申請提供包括所述抗-PCSK9抗體及其抗原結合片段的套組。在一些實施例中，所述套組用於偵測在生物樣品中的PCSK9的存在情況或水準。所述生物樣品可包括血清。在一些實施例中，所述套組包括與可偵測標記綴合的抗-PCSK9抗體及其抗原結合片段。在一些實施例中，所述套組包括未標記的抗-PCSK9抗體及其抗原結合片段，且進一步包括能夠與未標記的抗-PCSK9抗體及其抗原結合片段結合標記的二抗。所述套組可進一步包括使用說明及在套組中將各組件分隔開的包裝。 在一些實施例中，所述套組用於治療、預防或延遲由PCSK9介導的疾病或病況。在一些實施例中，所述抗-PCSK9抗體及其抗原結合片段與受質或儀器連接用於夾心測定如ELISA或免疫層析測定。適用的受質或儀器可為例如微孔板及試紙。 在一些實施例中，所述套組亦包括一或多種已知對降低膽固醇有益的試劑。例示性的試劑包括他汀類藥物、他汀類以外的HMG-CoA還原酶抑制劑，菸酸(尼克酸)、膽固醇吸收抑制劑、膽固醇酯轉移蛋白(CETP)、膽汁酸螯合劑、貝特類、植物甾醇；或選自小分子的脂質/脂質濃度比的調節劑、擬肽、反義RNA、小干擾RNA(siRNA)及天然或改性脂。在某些實施例中，膽固醇吸收抑制劑為依折麥布或SCH-48461；CETP為evacetrapib，anacetrapib或dalcetrapib；膽汁酸螯合劑較佳為考來維侖，消膽胺或貝特類降脂寧較佳為非諾貝特，吉非貝齊，安妥明，或苯札貝特；或上述試劑之組合。醫藥組合物及治療方法 本申請進一步提供包括所述抗-PCSK9抗體及其抗原結合片段的醫藥組合物及一或多個醫藥學上可接受之載體。 用於本申請揭示之醫藥組合物中的藥用可接受載劑可包括，例如，藥用可接受的液體、凝膠或固體載劑、水相介質、非水相介質、抗微生物物質、等滲物質、緩衝液、抗氧劑、麻醉劑、懸浮劑/分散劑、螯合劑、稀釋劑、佐劑、輔料或無毒輔助物質，其他此項技術公知的組分或以上的多種組合。 適用的組分可包括例如抗氧劑、填充劑、黏合劑、崩解劑、緩衝液、防腐劑、潤滑劑、攪味劑、增稠劑、著色劑、乳化劑或穩定劑，例如糖及環糊精。適用的抗氧劑可包括例如甲硫胺酸、抗壞血酸、EDTA、硫代硫酸鈉、鉑、過氧化氫酶、檸檬酸、半胱胺酸、巰基甘油、巰基乙酸、巰基山梨醇、丁基甲基茴香醚、丁基化羥基甲苯及/或沒食子酸丙酯。如本發明所揭示，在一種含有本發明揭示之抗體或其抗原結合片段之組合物中包括一或多種抗氧劑如甲硫胺酸，可將降低所述抗體或其抗原結合片段的氧化。對氧化作用的減少可防止或減少結合親和力的降低，從而提高抗體穩定性且延長保質期。因此，在某些實施例中，本發明提供之組合物中含有一或多種所述之抗體或其抗原結合片段以及一或多種抗氧劑，例如甲硫胺酸。本發明進一步提供多種方法，藉由將本發明中提供的抗體或其抗原結合片段與一或多種抗氧劑混合，例如甲硫胺酸，可防止所述抗體或其抗原結合片段氧化、延長其保質期及/或提高其活性。 進一步的說，藥用可接受的載劑可包括，例如，水相介質如氯化鈉注射液、林格氏液注射液、等滲葡萄糖注射液、無菌水注射液、或葡萄糖及乳酸林格注射液、非水介質例如：植物來源的不揮發性油、棉花子油、玉米油、芝麻油、或花生油、細菌抑制或真菌抑制濃度下的抗菌物質、等滲劑如：氯化鈉或葡萄糖、緩衝液如：磷酸鹽或枸櫞酸酸鹽緩衝液，抗氧化劑如：硫酸氫鈉，局部麻醉劑如：鹽酸普魯卡因，助懸劑及分散劑如：羧甲基纖維素鈉、羥丙基甲基纖維素或聚乙烯吡咯啶酮，乳化劑如：聚山梨醇酯80 (吐溫-80)、螯合試劑如EDTA (乙二胺四乙酸) 或EGTA (乙二醇雙(2-胺基乙基醚)四乙酸)、乙醇、聚乙二醇、丙二醇、氫氧化鈉、鹽酸、檸檬酸或乳酸。作為載劑的抗菌劑可加入多次劑量容器中的醫藥組合物中，其包括酚類或甲酚、汞製劑、苯甲醇、氯代丁醇、甲基及丙基對羥基苯甲酸酯、噻汞撒、氯苯甲烷銨及氯苯乙銨。適用的輔料可包括，例如，水、鹽、葡萄糖、甘油或乙醇。適用的無毒輔助物質可包括，例如，乳化劑、pH值緩衝劑、穩定劑、增溶劑，或醋酸鈉、去水山梨糖醇月桂酸酯、三乙醇胺油酸酯或環糊精之類的物質。 所述醫藥組合物可為液體溶液、懸浮液、乳劑、丸劑、膠囊、錠劑、持續釋放製劑或粉末。口服製劑可包括標準載體如藥物級的甘露醇、乳糖、澱粉、硬脂酸鎂、聚乙烯吡咯啶酮、糖精鈉、纖維素、碳酸鎂等。 在某些實施例中，所述醫藥組合物經製備為可注射組合物。可注射醫藥組合物可以任何習知之形式製備，例如，液體溶劑、懸浮劑、乳化劑或適用於產生液體溶劑、懸浮劑或乳化劑的固體形式。注射製劑可包括現用的無菌及/或無熱原溶液、使用前先與溶劑結合的無菌乾燥的可溶物，如凍乾粉，包括皮下片、注射即用的無菌懸浮劑、使用前先與介質結合的無菌乾燥不溶產品，及無菌及/或無熱原的乳劑。溶劑可為水相或非水相。 在某些實施例中，單位劑量的注射製劑包裝在一個安瓿、一支管或一支帶有針的針筒中。此項技術習知，所有注射給藥的製劑應為無菌無熱原。 在某些實施例中，藉由將本申請揭示之抗體或其抗原結合片段溶解於某適當的溶劑中可製備無菌凍乾的粉末。所述溶劑可含有一種可提高粉或由粉末製得的重組溶液的穩定性，或改善粉末或重組溶液的其他藥理組分。適用的輔料包括，但不限於，水、葡萄糖、三梨糖醇、果糖、玉米糖漿、木糖醇、甘油、葡萄糖、蔗糖或其他適用的物質。溶劑可含有緩衝液，如枸櫞酸緩衝液、磷酸鈉或磷酸鉀緩衝液或其他熟習此項技術者公知的緩衝液，在一個實施例中，緩衝液的pH為中性。在此項技術公知的標準條件下進行對所述溶解進行隨後的過濾除菌，然後凍乾製得理想的製劑。在一個實施例中，將所得的溶劑分裝至小管中凍乾。每支小管可容納單次劑量或多次劑量的所述抗-PCSK9抗體或其抗原結合片段或其組合物。每支小管中的裝入量可略微高於每次劑量所需或多次劑量所需(例如10%過量)，從而保證取樣精確及給藥精確。凍乾粉可在適當的條件下儲存，如在約4℃至室溫範圍。 用注射用水將凍乾粉重溶得到用於注射給藥的製劑。在一個實施例中，可將凍乾粉加至無菌無熱原水或其他適用的液體載劑中重溶。精確的量由選擇的療法決定，可根據經驗值決定。 亦提供治療方法，包括將治療有效量的本申請所述之抗體或其抗原結合片段施用給需要其之個體，由此治療或預防與PCSK9相關的病況或病症。在另一態樣中，亦提供治療將自上調的免疫響應獲益的個體病況之方法，包括對所述需要其之個體施用治療有效量的本申請所述之抗體或其抗原結合片段。 本申請中提供的抗體或其抗原結合片段的治療有效劑量依賴於此項技術公知的多種因素，例如體重、年齡、過往病史、現用治療、對象的健康病況及交叉感染的潛力、過敏、超敏及副作用，以及給藥途徑及腫瘤發展的程度。熟習此項技術者(例如醫生或獸醫)可根據此等或其他條件或要求按比例降低或升高劑量。 在某些實施例中，本發明提供的抗體或其抗原結合片段可在治療有效劑量約0.01 mg/kg至約100 mg/kg之間給藥(例如，約0.01 mg/kg、約0.5 mg/kg、約1 mg/kg、約2 mg/kg、約3mg/kg、約5 mg/kg、約10 mg/kg、約15 mg/kg、約20 mg/kg、約25 mg/kg、約30 mg/kg、約35 mg/kg、約40 mg/kg、約45 mg/kg、約50 mg/kg、約55 mg/kg、約60 mg/kg、約65 mg/kg、約70 mg/kg、約75 mg/kg、約80 mg/kg、約85 mg/kg、約90 mg/kg、約95 mg/kg或約100 mg/kg)。在某些實施例中，所述抗體或其抗原結合片段以約50 mg/kg或更少的劑量給藥，在某些實施例中，給藥劑量為10 mg/kg或更少、5 mg/kg或更少、3 mg/kg或更少、1 mg/kg或更少、0.5 mg/kg或更少或0.1 mg/kg或更少。某特定劑量可在多個間隔給藥，例如每天一次、每天兩次或更多、每月兩次或更多、每週一次、每兩週一次、每三週一次、每月一次或每兩月或更多月一次。在某些實施例中，給藥劑量可隨治療進程變化。例如，在某些實施例中，初始給藥劑量可比後續給藥劑量高。在某些實施例中，給藥劑量在治療進程中根據給藥對象的反應進行調整。 給藥方案可藉由調整達到最優反應(如治療反應)。例如，可進行單劑量給藥或在一段時間分多個分隔的劑量給藥。 本發明中揭示之抗體及抗原結合片段可藉由此項技術公知的給藥方式給藥，例如注射給藥(如，皮下注射、腹腔注射、靜脈注射，包括靜脈滴注，肌肉注射或皮內注射)或非注射給藥(如，口服給藥、鼻腔給藥、舌下給藥、直腸給藥或外用給藥)。 使用方法 本申請進一步提供使用所述抗-PCSK9抗體或其抗原結合片段之方法。 在一些實施例中，本申請提供在個體中治療PCSK9介導的病況或病症之方法，包括施用治療有效量的本申請所述之PCSK9抗體或其抗原結合片段。在一些實施例中，所述個體經鑑定為患有可能對PCSK9抑制劑響應之病症或病況。在某些實施例中，所述個體處於具有或發展由PCSK9介導的疾病或病況的風險，所述PCSK9介導的疾病或病況表現出一或多種所述疾病或病況的症狀，如超重、具有升高的膽固醇水準、具有編碼LDL-R或APOB的基因的遺傳性突變或具有此類疾病或病況的家族病史。在某些實施例中，所述個體在治療中對另一種降低膽固醇的試劑(例如，他汀類藥物)為抵抗的或不耐受的，因此在該治療中膽固醇水準無法有效地降低至可接受的水準。在某些實施例中，所述由PCSK9介導之疾病或病況包括感染性疾病，如嚴重蜂窩織炎、腸胃炎、敗血症、肺炎、皮膚及軟組織感染、腎盂腎炎、病毒感染，例如，乙型肝炎、丙型肝炎、疱疹病毒的病毒感染、Epstein-Barr病毒、艾滋病毒、巨細胞病毒、單純疱疹病毒I型、單純疱疹病毒2型、人乳頭狀瘤病毒、腺病毒、卡波西西肉瘤相關疱疹病毒流行病、薄環病毒(Torquetenovirus)、JC病毒或BK病毒，或包括炎性疾病，如阿茲海默氏症、強直性脊柱炎、關節炎(骨關節炎、類風濕關節炎(RA)、牛皮癬性關節炎)、哮喘、動脈粥樣硬化、克羅恩病、結腸炎、皮炎、憩室炎、纖維肌痛、肝炎、腸易激症候群(IBS)、系統性紅斑狼瘡(SLE)、腎炎、帕金森氏病及潰瘍性結腸炎。 在目標生物組織中LDL-C的存在情況及水準可指示所述生物樣品來源的個體是否可能對PCSK9抑制劑響應。可使用多種方法在來自所述個體的待測生物樣品中確定LDL-C的存在情況或水準。在美國使用毫克(mg)每分升(dL)血液量測膽固醇水準，而在加拿大及許多歐洲國家則使用毫莫耳(mmol)每公升(L)血液量測。 在一些實施例中，在所述待測生物樣品中LDL-C、總膽固醇或非HDL-C的存在或水準上調表示響應的可能性。本申請使用的術語「上調」係指與使用相同抗體偵測的參照樣品中膽固醇水準相比，使用本申請所述之抗體或其抗原結合片段在待測樣品中偵測的膽固醇水準的總的增加不少於10%、15%、20%、25%、30%、35%、40%、45%、50%、55%、60%、65%、70%、75%、80%或更多。所述參照樣品可為自健康或無疾病的個體中獲得的對照樣品，或自待測樣品來源的個體中獲得的健康或無疾病的樣品。 本發明揭示之抗體及抗原結合片段可單獨給藥或與一或多種其他治療手段或物質聯合給藥。例如，本發明揭示之抗體及抗原結合片段可與他汀類藥物、他汀類以外的HMG-CoA還原酶抑制劑，菸酸(尼克酸)、膽固醇吸收抑制劑、膽固醇酯轉移蛋白(CETP)、膽汁酸螯合劑、貝特類、植物甾醇；或選自小分子的脂質/脂質濃度比的調節劑、擬肽、反義RNA、小干擾RNA(siRNA)及天然或改性脂。在某些實施例中，膽固醇吸收抑制劑為依折麥布或SCH-48461；CETP為evacetrapib，anacetrapib或dalcetrapib；膽汁酸螯合劑較佳為考來維侖，消膽胺或貝特類降脂寧較佳為非諾貝特，吉非貝齊，安妥明，或苯札貝特；或上述試劑之組合進行聯用。 在某些此類實施例中，本發明揭示之抗體及抗原結合片段與一或多種上述治療物質聯用時，可與所述之一或多種治療物質同時給藥，在某些此類實施例中，所述之抗體及抗原結合片段可作為同一個醫藥組合物的一部分同時給藥。但是，與其他治療物質「聯用」的抗體及抗原結合物不需要同時給藥或與該治療物質在同一組合物中給藥。本發明中「聯用」的含義亦包括在另一個治療物質之前或之後給藥的抗體及抗原結合物亦視為與該治療物質「聯用」，即使所述抗體或其抗原結合片段與第二種物質藉由不同給藥方式給藥。在可能的情況下，與本發明揭示之抗體或其抗原結合片段聯用的其他治療物質可參照該其他治療物質的產品說明書之方法用藥，或參照外科醫生的案頭參考書2003(Physicians' Desk Reference，第57版; Medical Economics Company; ISBN: 1563634457; 第57版 (2002年11月))，或參照其他此項技術公知之方法。 以下實施例旨在更好地說明本發明，且不應理解為限制本發明的範圍。所有下述的特定組合物、材料及方法，其整體或部分，均在本發明的範圍內。此等特定組合物、材料及方法不是為了限制本發明，而只是為說明特定的實施例在本發明的範圍內。熟習此項技術者可不添加創造性及不偏離本發明範圍而開發出等同的組合物、材料及方法。應理解，在對本發明之方法作出之多種改動仍可包括在本發明範圍內。發明人意在將此類變動包括在本發明的範圍內。實施例 1 ：抗原及其他蛋白的產生 1.1人源及鼠源PCSK9 將人及鼠的PCSK9基因分別組裝至pcDNA3.3載體，C端融合了6-His標籤或鼠源Fc標籤。用轉染試劑PlasFect (Bioline USA, BIO-46026)，將此等質粒分別轉染至HEK293細胞。轉染後收集細胞上清。His標籤蛋白藉由Ni柱(Qiagen Inc)純化，鼠源Fc融合蛋白藉由Protein A管柱(MabSelect SuRe, GE)純化。 1.2人源 LDL-R 將LDL-R胞外區基因組裝至pcDNA3.3載體，C端融合了6-His標籤。用轉染試劑PlasFect (Bioline USA, BIO-46026)，將此等質粒轉染至HEK293細胞。轉染後收集細胞上清，LDL-R蛋白第一步用Ni 柱(Qiagen Inc) 純化，第二步用離子交換管柱純化。 1.3參照抗體 參照抗體BMK.115的序列為基於美國專利號8889834B2中的21B12序列。將含有重鏈及輕鏈的質粒共轉染至HEK293細胞。轉染後收集細胞上清，抗體藉由Protein A管柱(MabSelect SuRe, GE) 純化。實施例 2 ：抗體的產生 2.1免疫 用含有人源免疫球蛋白可變區基因的轉殖基因大鼠OmniRat® (OMT)進行免疫以獲得全人抗體。人源PCSK9蛋白足底注射，每三天一次。6次免疫後進行第一次效價偵測，之後每週免疫一次。 2.2血清效價偵測 使用酶聯免疫吸附法(ELISA)偵測大鼠血清效價。先用pH值9.2的包被液將人源PCSK9蛋白稀釋至1 μg/ml，加入至酶標板(Nunc)中，4℃隔夜培育。洗板後封閉，每孔加入200 μl封閉液1× PBS/ 2% BSA，室溫靜置培育1小時。起始孔加入稀釋100倍的大鼠血清，然後用封閉液以3倍的比例進行連續梯度稀釋，室溫靜置培育1小時。洗板後加入山羊抗大鼠IgG1及山羊抗大鼠IgG2b的HRP酶標二抗混合物(Bethyl)，室溫靜置培育1小時。洗板後加入TMB受質顯色液，然後用2M 鹽酸終止顯色。使用酶標儀(Molecular Device)讀取在450 nM處的吸光值。 2.3動物免疫與雜交瘤的產生 選取滴度達到要求的大鼠，取出淋巴結及脾臟，轉移至組織研磨器中研磨。100目篩網過濾後計數B細胞。骨髓瘤P3細胞用培養基調整至適當的體積後計數。將兩種細胞按照B細胞:P3 = 1:1數目重懸且混勻。細胞懸浮液加入融合槽內進行電融合 (BTX ECM2001). 融合後將細胞轉移至含有1/2 HA培養基中，按每板5×10⁵ 個細胞的密度鋪板。 免疫後的動物血清中抗原特異性抗體的滴度用ELISA進行偵測。挑選出滴度達到312500的大鼠進行融合產生雜交瘤細胞。 2.4雜交瘤篩選 ELISA法偵測結合：先用pH值9.2的包被液將鏈黴親和素(Streptavidin)稀釋至1 μg/ml，加入至96孔酶標板中(Nunc)，4℃隔夜培育。封閉洗板後加入生物素標記的人源PCSK9蛋白，濃度為250 ng/ml，室溫靜置培育1小時。洗板後加入雜交瘤細胞上清，室溫靜置培育1小時。洗板後加入山羊抗大鼠IgG1及山羊抗大鼠IgG2b的HRP酶標二抗 (Bethyl)混合物，室溫培育45 分鐘。洗板後加入TMB受質顯色液，用2M 鹽酸終止顯色，然後使用酶標儀(Molecular Device)讀取在450奈米處的吸光值。 競爭ELISA：酶標板(Nunc)中加入LDL-R，4℃隔夜培育。同時將雜交瘤細胞上清與生物素標記的人PCSK9蛋白混合，PCSK9蛋白的終濃度為250 ng/ml，4℃隔夜培育。酶標板洗板封閉後，加入雜交瘤細胞上清與PCSK9蛋白的混合物，室溫靜置培育1小時。洗板後加入HRP標記的鏈黴親和素。最後加入TMB受質顯色液，且用2 M鹽酸終止顯色，然後使用酶標儀(Molecular Device)讀取在450奈米處的吸光值。 抗體篩選 首輪篩選用雜交瘤培養上清進行抗原結合偵測。4次融合共篩選出13000個與抗原有特異性結合的雜交瘤。對此等雜交瘤細胞株進行進一步的競爭實驗的篩選。藉由競爭ELISA的篩選得到104株雜交瘤細胞，其分泌的抗體可阻斷人源PCSK9與人源LDL-R的結合。將此等能夠結合抗原併有阻斷功能的抗體自雜交瘤細胞上清中純化出來。同時對此等雜交瘤細胞株進行次選殖。次選殖用結合及競爭ELISA進行偵測，且對其亞型進行鑑定。 純化後的抗體進行細胞LDL吸收實驗。結合與阻斷活性亦將藉由純化的抗體進行進一步偵測。最終的候選純系係根據結合的親和力，阻斷PCSK9結合LDL-R的能力以及恢復細胞LDL吸收的能力選定。 2.5雜交瘤細胞次選殖 將選取的雜交瘤細胞株按照每孔0.5個、1個及5個細胞的密度鋪至96孔板中。選出其中的單株孔用ELISA進行偵測。各雜交瘤細胞株保留且凍存3個次純系。 2.6抗體亞型偵測 用ELISA鑑定抗體同種型(見表2)。分別用包被液將山羊抗大鼠IgG1、IgG2a、IgG2b、IgG2c、IgM的抗體(Bethyl)稀釋至1 μg/ml，加入至96孔酶標板(Nunc)中，4℃隔夜培育。洗板封閉後，加入雜交瘤細胞培養上清，室溫靜置培育1小時。洗板後分別加入山羊抗人Kappa鏈酶標二抗(Southern Biotech)，或山羊抗人Lambda鏈酶標二抗(Southern Biotech)，室溫靜置培育45 分鐘。洗板後加入TMB受質顯色液，用2 M鹽酸終止顯色後使用酶標儀(Molecular Device)讀取在450奈米處的吸光值。表 2. 抗體亞型

2.7抗體純化 將收集的雜交瘤上清調節pH至7.0後，上樣至Protein A管柱 (MabSelect SuRe, GE)。抗體用Glycine洗脫，且立即用1M Tris中和。純化後的蛋白濃度用Nano Drop (Thermal-Fisher)偵測。蛋白的純度藉由SDS-PAGE (Invitrogen, NuPAGE4%-12% Bis-Tris Gel) 及HPLC-SEC (Agilent)偵測。實施例 3 ：全人源抗體的產生 3.1 雜交瘤測序 採用Trizol套組(Invitrogen-15596018)自雜交瘤細胞中提取RNA，再利用5'-RACE套組(Takara-28001488)擴增得到cDNA，然後藉由3'-簡併引物及3'-接頭引物(ExTaq: Takara-RR001B)擴增cDNA。將擴增得到的片段插入至pMD18-T載體(Takara-D101C)且送測序(上海鉑尚公司)。 選擇的抗體11.4、18.156.8、15.14.2、17.72.3、18.136.7、19.3.8及40409的可變區序列(胺基酸序列及核酸序列)如SEQ ID NO: 73-100所示。 3.2全人源抗體的表現與純化 將各抗體(人-鼠嵌合抗體)可變區基因序列選殖至含有人源恆定區基因的pcDNA3.3載體上。將重鏈及輕鏈質粒轉染至HEK293細胞中進行抗體表現。離心收集細胞上清。用Protein A (MabSelect SuRe, GE)純化抗體，且將純化後的抗體透析至PBS中。抗體濃度藉由Nanodrop。蛋白的純度藉由SDS-PAGE (Invitrogen, NuPAGE4%-12% Bis-Tris Gel)及HPLC-SEC (Agilent)偵測。 3.3親和力成熟 構建11.4 重鏈CDR3區的飽和突變文庫，且用ELISA方法進行篩選。挑選與原始純系相比，單點突變使PCSK9親和力提高之純系。將挑選出的突變位點建成一個組合文庫。根據ELISA 結合實驗及 SPR k_off 排序結果，挑選出親和力提高的突變組合。 以11.4序列作為模板，構建2個隨機突變文庫。藉由對該文庫進行2輪淘選及篩選，挑選出能夠提高親和力的突變，且與飽和突變文庫中得到的突變組合，從而進一步提高抗體的親和力。 構建11.4 重鏈CDR3區的飽和突變文庫，且用ELISA方法進行篩選。與原始純系相比，單點突變挑選出PCSK9親和力提高的純系(圖1)。將挑選出的突變位點建成一個組合文庫。根據ELISA結合實驗及SPR k_off 排序結果，挑選出親和力提高的突變組合。CDR3區的3個位點的突變組合可使親和力提高10倍，如純系B4G2及C1B4(見表3)。 表3. 親和力成熟後的抗體的親和力

以11.4序列作為模板，構建2個隨機突變文庫。藉由對該文庫進行2輪淘選及篩選，得到5個能夠提高親和力的突變。將其中3個突變與B4G2中的突變組合，得到三個純系40408、40409及40410，從而使親和力進一步提高2倍。最終親和力成熟後的抗體40409的親和力比參照抗體BMK.115高2倍 (見表4)。 表4. 親和力成熟後的抗體的親和力

3.4瞬轉表現全人抗體 3.4.1 18.156.8 (hIgG4) 全人抗體18.156.8-hIgG4)在還原條件下的SDS-PAGE上的表徵分子量為25 kDa 及55 kDa，分別對應抗體的輕鏈及重鏈。在非還原SDS-PAGE上的主條帶對應了完整的IgG分子，分子量約150 KD。HPLC-SEC結果顯示抗體純度為99.6% (圖3)。內毒素含量小於0.5 EU/mg。 3.4.2 40409 (hIgG4) 全人抗體WBP301-40409 (hIgG4)在還原條件下的SDS-PAGE上的表徵分子量為25 kDa 及55 kDa，分別對應抗體的輕鏈及重鏈。在非還原SDS-PAGE上的主條帶對應了完整的IgG分子，分子量約150 KD。HPLC-SEC結果顯示抗體純度為98%(圖5)。內毒素含量小於0.5 EU/mg。 3.4.3 15.14.2-uAb-IgG4L 全人抗體15.14.2-uAb-IgG4L在還原條件下的SDS-PAGE上的表徵分子量為25 kDa 及55 kDa，分別對應抗體的輕鏈及重鏈。在非還原SDS-PAGE上的主條帶對應了完整的IgG分子，分子量約150 KD。HPLC-SEC結果顯示抗體純度為99.8% (圖7)。內毒素含量小於0.5 EU/mg。 3.4.4 17.72.3-uAb2-IgG4K 全人抗體17.72.3-uAb2-IgG4K在還原條件下的SDS-PAGE上的表徵分子量為25 kDa 及55 kDa，分別對應抗體的輕鏈及重鏈。在非還原SDS-PAGE上的主條帶對應了完整的IgG分子，分子量約150 KD。HPLC-SEC結果顯示抗體純度為98.9%(圖9)。內毒素含量小於0.5 EU/mg。 3.4.5 18.136.7-IgG4K 全人抗體18.136.7-IgG4K在還原條件下的SDS-PAGE上的表徵分子量為25 kDa 及55 kDa，分別對應抗體的輕鏈及重鏈。在非還原SDS-PAGE上的主條帶對應了完整的IgG分子，分子量約150 KD。HPLC-SEC結果顯示抗體純度為99.2% (圖11)。內毒素含量小於0.5 EU/mg。 3.4.6 19.3.8-uAb1-IgG4L 全人抗體19.3.8-uAb1-IgG4L在還原條件下的SDS-PAGE上的表徵分子量為25 kDa 及55 kDa，分別對應抗體的輕鏈及重鏈。在非還原SDS-PAGE上的主條帶對應了完整的IgG分子，分子量約150 KD。HPLC-SEC結果顯示抗體純度為99.9% (圖13)。內毒素含量小於0.5 EU/mg。實施例 4 ：候選抗體表徵 4.1全人抗體的結合與阻斷活性 ELISA方法驗證全人抗體與PCSK9結合的活性以及阻斷PCSK9與LDL-R結合的活性分別見圖14及15。結合實驗EC50與阻斷實驗的IC50結果見表5及表6。候選抗18.156.8(hIgG4)、40409(hIgG4)、15.14.2-uAb-IgG4L及17.72.3-uAb2-IgG4K顯示與參照抗體BMK.115及Repatha相當的結合與阻斷活性。表 5. 抗體與PCSK9結合活性

表 6. 阻斷活性

4.2 LDL 吸收實驗： HepG2或Huh-7細胞用含10% FBS的DMEM培養液以每孔1×10⁵ 的密度接種於96孔板，置於37℃培養箱中。次日將含10% FBS的DMEM培養液換成無血清培養液，且將野生型PCSK9或變體PCSK9 (D374Y) 與一系列濃度梯度稀釋的抗體混合後加入相應的孔中，37℃培育1小時。野生型PCSK9及變體PCSK9 (D374Y) 的終濃度分別為20μg/ml及1.3μg/ml。向96孔板中加入Bodipy螢光標記的LDL (Invitrogen L-3483)，終濃度為1.5μg/ml。在37℃培養箱中培育3小時後取出96孔板，棄去含有LDL的培養液，用胰酶消化收集細胞且洗滌兩次。細胞內的螢光用FACS偵測，螢光強度表徵了LDL的吸收量。LDL吸收的回復率由如下公式計算：LDL吸收回復率 (%) = (MFI_樣品 - MFI_LDL+Ag1H )/ (MFI_{LDL - MFILDL+Ag1H} ) ×100% 。 在野生型及突變體PCSK9存在情況下，在HepG2及Huh-7細胞上對抗體18.156.8 及40409進行LDL吸收活性的實驗(見圖16及17)，IC50值見表7。實驗證明無論在有野生型或突變型PCSK9存在的條件下，抗體18.156.8 及40409均能有效地恢復HepG2及Huh-7細胞對LDL的吸收能力。在野生型PCSK9存在情況下，在HepG2細胞上對抗體15.14.2, 17.72.3及19.3.8進行LDL吸收活性的實驗 (圖18)， IC50值見表8. 實驗證明在有野生型PCSK9存在的條件下，抗體15.14.2, 17.72.3及19.3.8能有效地恢復HepG2細胞對LDL的吸收能力。 表7. 抗體恢復HepG2及Huh-7細胞LDL吸收的IC50

表8. 抗體恢復HepG2細胞LDL吸收的IC50

4.3動力學親和力 4.3.1運用SPR技術偵測結合動力學常數 使用Biacore T200 (GE) 偵測抗體與人源PCSK9及恆河猴PCSK9的結合親和力常數。使用Protein A或抗IgG Fc抗體偶聯的芯片補貨待測抗體。然後將不同濃度的人源PCSK9或恆河猴PCSK9分別注入傳感器芯片上，進樣速度為30μl/min。樣品結合時間為180 s, 解離時間為1200 s。每次抗原結合後，用2 M MgCl₂ 再生芯片。 最終結合解離曲線為扣除參比通道Fc1及緩衝液通道信號後的結果。實驗資料用1：1 Langmiur模式進行擬合。計算PCSK9莫耳濃度時所用分子量為85 KDa。 4.3.2與恆河猴PCSK9蛋白結合反應 用pH值9.2的包被液將小鼠抗His標籤的抗體(Genscript)稀釋至1 μg/ml，加入至96孔酶標板中，4℃隔夜培育。洗板封閉後，加入恆河猴PCSK9-His (Sino Biological)蛋白，濃度為1 μg/ml，室溫培育1小時。洗板後加入待測抗體，室溫培育1小時。洗板後加入山羊抗大鼠IgG1及山羊抗大鼠IgGb的HRP酶標二抗 (Bethyl)混合物，室溫培育45 分鐘。最後加入TMB受質顯色液，反應後用2M 鹽酸終止顯色，然後使用酶標儀(Molecular Device)讀取在450奈米處的吸光值。 用SPR實驗偵測抗體的動力學結合常數。抗體與人源PCSK9及猴源PCSK9結合的親和力見表9。 表9. 抗體與人源PCSK9及猴源PCSK9結合之動力學常數

4.4抗體在血清中之穩定性偵測 待測抗體用新鮮分離的人血清稀釋(血清含量＞95%)後，置於37℃培養箱中分別培育0、1、3、7、14天。在各時間終點處，自37℃培養箱中取出樣品後，在乙醇-乾冰浴中迅速冷凍且將樣品置於-80℃冰箱保存。穩定性測試前取出樣品迅速溶解。96孔酶標板用Na₂ CO₃ /NaHCO₃ (pH 9.2)緩衝液稀釋的鏈黴親和素包被，置於4℃冰箱。次日，96孔板用0.1% PBST洗滌且用2% BSA/PBS封閉1小時後，加入生物素標記的PCSK9。在室溫下培育1小時後洗滌，加入一系列濃度梯度稀釋的樣品。在室溫下培育1小時後洗滌，加入HRP標記的羊抗人IgG抗體。在室溫下培育1小時後洗滌，加入TMB受質，待顯色後用2M HCl終止。用酶標儀(Molecular Device)讀取波長450nm處的吸光度。 抗體在37度人血清中培育後，用ELISA偵測其與PCSK9結合的活性(見圖19)。抗體18.156.8 及B4G2在血清中培育1天，3天，7天及14天後，結合活性與未培育的抗體相同，說明18.156.8及B4G2在37度可穩定存在於人血清中至少14天。抗體15.14.2, 17.72.3 及19.3.8在血清中培育3天後，結合活性與未培育的抗體相同。說明抗體可穩定存在於血清中至少3天。實施例 5 ：動物實驗 5.1食蟹猴中單次注射藥效 使用LDLC及HDLC3套組(Roche)在Roche/Hitachi cobas c系統上偵測猴子血清中低密度脂蛋白膽固醇LDL-C及高密度脂蛋白膽固醇HDL-C的濃度。總膽固醇TCHO用cholesterol FS 套組 (DiaSys)偵測。 5.1.1實驗1: 6隻3-4歲的雌性食蟹猴，體重在2.6至2.9公斤之間，隨機分為6組(每組1隻)。6組猴子分別進行單次靜脈注射10 mg/kg或30 mg/kg劑量之BMK.115或18.156.8 (hIgG4)或40409 (hIgG4)抗體。給藥當天定義為第一天。每組猴子給藥後觀測36天。

備註：在本實驗中，「劑量水準」及「劑量」互換使用。^a 除非另有說明書，否則劑量代表活性成分。 抗體18.156.8 及40409在食蟹猴中降低低密度膽固醇的療效。食蟹猴分別按10 mg/kg及30 mg/kg的劑量給藥後，抗體BMK.115 及W301-18.156.8可快速且持久地降低低密度脂蛋白膽固醇及總膽固醇含量。 在10 mg/kg及30 mg/kg的劑量組中，與注射前相比，BMK.115分別使低密度膽固醇降低至83.4% 及 89.6%；18.156.8分別使低密度膽固醇降低至79.5% 及83.5%。最大降低量在第8天達到。直至偵測結束，無論10 mg/kg或30 mg/kg劑量組，注射了18.156.8的猴子的低密度膽固醇一直維持在很低水準；對照抗體BMK.115隻在30 mg/kg劑量組中可使低密度脂蛋白水準維持在低水準至36天，但在10 mg/kg劑量組中，低密度脂蛋白水準自第24天開始回升，到第28天已恢復至給藥前水準 (圖20A及20B)。所以與參照抗體BMK.115相比，18.156.8可更長時間地將低密度脂蛋白維持在較低水準。 在10 mg/kg 及30 mg/kg的劑量組中，與注射前相比，40409 (hIgG4)分別使低密度膽固醇降低至32.2%及38.1%(圖20A及20B)。 18.156.8 (hIgG4)及40409 (hIgG4)抗體給藥的猴子中，無論10 mg/kg或30 mg/kg劑量組，高密度脂蛋白膽固醇水準在實驗過程中均沒有明顯變化。參照抗體BMK.115給藥的猴子中，10 mg/kg劑量組的猴子的高密度膽固醇水準無明顯變化，但30 mg/kg劑量組的高密度膽固醇水準與給要前相比降低了30% (圖21A及21B)。 5.1.2實驗2：10隻3-4歲的雌性食蟹猴，體重在2.5至3.5公斤之間，隨機分為10組(每組1隻)。10組猴子分別進行單次靜脈注射3 mg/kg或10 mg/kg劑量的Repatha或15.14.2、17.72.3、18.136.7或19.3.8抗體。給藥當天定義為第一天。每組猴子給藥後觀測36天。

備註：在本實驗中，「劑量水準」及「劑量」互換使用。^a 除非另有說明書，否則劑量代表活性成分。 抗體15.14.2、17.72.3、18.136.7及19.3.8在食蟹猴中降低低密度膽固醇療效。 食蟹猴分別按3 mg/kg 及10 mg/kg的劑量給藥後，抗體15.14.2及Repatha可快速且持久地降低低密度脂蛋白膽固醇及總膽固醇含量(圖22A及22B)。抗體18.136.7在3 mg/kg及10 mg/kg劑量組中，亦使低密度膽固醇有顯著降低(~50%)。17.72.3及19.3.8在10 mg/kg劑量組中使低密度膽固醇有少量降低。 在3 mg/kg及10 mg/kg的劑量組中，與給藥前相比，Repatha分別使低密度膽固醇降低至80%及77%；15.14.2在兩個劑量組均使低密度膽固醇降低至77%。最大降低量在第8-16天到達。在10 mg/kg劑量組中，注射了15.14.2的猴子的低密度膽固醇一直維持在很低水準，直至偵測結束。而對照抗體Repatha給藥的猴子的低密度脂蛋白水準自第24天開始回升，到第36天已恢復至給藥前水準(圖22A及22B)。在3 mg/kg劑量組中，Repatha給藥的猴子的低密度脂蛋白水準第12天開始回升至80%，到第20天已恢復至給藥前水準。15.14.2給藥的猴子到第28天低密度膽固醇水準仍維持在給藥前50%以下。所以與Repatha相比，在3 mg/kg 及10 mg/kg兩個劑量組中，15.14.2均可更長時間地將低密度脂蛋白維持在較低水準。 高密度脂蛋白膽固醇在所有給藥的猴子中均沒有明顯變化(圖23 A及23B)。 5.2藥代動力學 藉由測定動物血清中的BMK.115、18.156.8、40409、15.14.2、17.72.3、18.136.7、19.3.8濃度以獲得體內藥物暴露水準。自所有存活的食蟹猴採集0小時(給藥前)，及給藥後0.5、1、2、4、24、48、96、168、336、504、672、744及840小時的血樣。 自動物的頭靜脈或股靜脈採集約2mL全血，置於無抗凝劑的採血管中。血樣採集後常溫靜置至少30分鐘。約4℃下2000 g離心10分鐘獲取血清(血樣採集後2小時內完成)。血清轉移至貼有標籤的聚丙烯樣品管中。立即垂直放入乾冰中速凍，然後保存在 ≤-60°的超低溫冰箱中。 在藥代動力學偵測前，將血清樣品迅速解凍。用Na₂ CO₃ /NaHCO₃ 緩衝液稀釋山羊抗人多株抗體後，包被酶標偵測板，4℃隔夜培育。用0.1% Tween-PBS洗板後，加入2% BSA/PBS封閉。將稀釋後的食蟹猴血清樣品加入酶標板中，於室溫下培育一小時。洗板後，先後於酶標板中加入生物素標記的山羊抗人IgG抗體及鏈黴親和素-HRP，分別於室溫下培育一小時。加入TMB受質，待顯色後用2M HCl終止。在450nm波長下讀取每孔吸光值。藉由標準曲線計算血清中抗體的濃度。 藥代動力學參數包括但不侷限於初始血清濃度(C₀ )及自0小時至給藥後840小時的血藥濃度-時間曲線下的面積(AUC_0-840h )，用驗證過的WinNonlin程序(PharsightVersion 6.2.1)進行計算。用非房室法的線性上升/對數下降梯形法則，計算AUC_0-840h ，限於對供試品處理的動物。在計算平均值時，將BLQ(低於定量值)當成0來計算。 血清中抗體的濃度用ELISA偵測(圖24及25)。由0至840小時血清中抗體濃度計算得到的BMK.115, 18.156.8(hIgG4)、40409(hIgG4)的C₀ 值及AUC_{0 - 840h} ，以及抗體半衰期見表10。 劑量自10 mg/kg增加至30 mg/kg時，18.156.8及40409的藥物暴露水準(AUC_{0 - 840h} 及/或C₀ )成比例增加，但BMK.115的藥物暴露水準增加與劑量的增加不成正比。 表10.藥代動力學資料I

由0至840小時血清中抗體濃度計算得到的Repatha、15.14.2、17.72.3、18.136.7及19.3.8的C₀ 值及AUC_{0 - 840h} ，以及抗體半衰期見表11。在3 mg/kg及10 mg/kg兩個劑量組，抗體15.14.2、17.72.3、18.136.7及19.3.8的半衰期均長於Repatha。 劑量自10 mg/kg增加至30 mg/kg時，Repatha、15.14.2、17.72.3、18.136.7及19.3.8的藥物暴露水準(AUC_{0 - 840h} 及/或C₀ )均成比例增加。 表11. 藥代動力學資料II

5.3免疫原性 自動物的頭靜脈或股靜脈採集0小時(給藥前), 及給藥後336、672、840小時的血樣。 用Na₂ CO₃ /NaHCO₃ 緩衝液稀釋抗體115、18.156.8及40409後鋪板(NUNC)，4度培育隔夜。用0.1% Tween-PBS洗板後加入2% BSA/PBS封閉。將PBS稀釋的猴子血清加入酶標板中室溫培育1小時。洗板後加入山羊抗食蟹猴IgG-HRP(與人IgG無交叉反應)培育。洗板後加入TMB受質，待顯色後用2M HCl終止。在450nm波長下讀取每孔吸光值。 抗體BMK.115, 18.156.8 (hIgG4) 及40409 (hIgG4)的免疫原性偵測結果見圖26。在給藥後336、672、840小時的猴子血清中，針對BMK.115、18.156.8 (hIgG4)、40409 (hIgG4) 的抗抗體的滴度與給藥前沒有明顯差異，說明單次注射10 mg/ml或30 mg/ml劑量的BMK.115、18.156.8 (hIgG4)或40409 (hIgG4) 抗體引起的免疫原性很低。 抗體Repatha、15.14.2、17.72.3、18.136.7及19.3.8的免疫原性偵測結果見圖27。在給藥後336、672、840小時的猴子血清中，針對Repatha、15.14.2、17.72.3、18.136.7、19.3.8的抗抗體的滴度與給藥前無明顯差異，說明單次注射10 mg/ml或30 mg/ml劑量之Repatha、15.14.2、17.72.3、18.136.7或19.3.8抗體引起的免疫原性很低。 5.4毒性 死亡/瀕死: 實驗期間每天2次報導各動物之健康狀態，上午1次下午1次，除動物到達設施及解剖當天動物檢查1次。 在整個實驗過程中動物無計劃外死亡。 詳細臨床觀察：對所有動物(包括替代動物)在實驗前進行1次詳細臨床觀察，對所有實驗動物在給藥日(約給藥後2±0.5小時)進行1次觀察，此後實驗過程中每週進行1次詳細臨床觀察。 整個實驗過程中未觀察到藥物相關的臨床症狀。 籠邊觀察：在實驗前期，自給藥前2天開始對所有動物(包括替代動物)每天進行1次籠邊觀察。在實驗第一天進行1次，在給藥日進行每天2次籠邊觀察(給藥後30分鐘以內及給藥後約6±0.5小時)，恢復期每天進行1次籠邊觀察。在相同時間段安排了詳細臨床觀察，未進行籠邊觀察。 體重：每隻動物實驗前期稱重1次。對實驗動物，在實驗第一天給藥前進行1次體重稱量，隨後實驗過程中每週稱重一次。 未觀察到藥物相關的體重變化，所有體重變化均在正常生物學差異範圍內。 攝食量：對所有動物，在實驗給藥前2天及整個實驗給藥及觀察過程中，評估動物的攝食量。藉由監視所有動物的食慾(動物是否進食構成評估文件)來評估每天的攝食量。 無藥物相關的攝食量變化。實施例 6 ：抗體 18.156.8 與參照抗體之間的活性比較 1. 參照抗體的產生 用實施例1及實施例2中所述之方法，基於專利CN101932607中12H11.1及24B9.1的序列產生參照抗體12H11.1.uIgG4K及24B9.1.uIgG4L。 抗體12H11.1.uIgG4K及24B9.1.uIgG4L在還原條件下的SDS-PAGE上的表徵分子量為25 kDa及55 kDa，分別對應抗體的輕鏈及重鏈(圖28A)。在非還原SDS-PAGE上的主條帶對應了完整的IgG分子，分子量約150 KD。HPLC-SEC結果顯示24B9.1.uIgG4L的抗體純度為96.8%(圖28B)。HPLC-SEC結果顯示12H11.1.uIgG4K的抗體純度為95.3%(圖28C)。 2. ELISA測定的與人PCSK9的結合 根據實施例2第2.4節雜交瘤篩選中所述之方法，用ELISA方法驗證參照抗體12H11.1.uIgG4K及24B9.1.uIgG4L與人PCSK9結合的活性(圖29)。結合EC50值見表12。參照抗體12H11.1.uIgG4K顯示低於抗體18.156.8及BMK.115的結合活性。參照抗體24B9.1.uIgG4L不與人PCSK9結合。 表12. 結合活性

3. ELISA法偵測阻斷 根據實施例2第2.4節雜交瘤篩選中所述之方法，藉由競爭ELISA偵測參照抗體12H11.1.uIgG4K及24B9.1.uIgG4L阻斷PCSK9與LDLR結合的活性(圖30)。IC50值見表13。抗體24B9.1.uIgG4L未顯示出阻斷活性。抗體12H11.1.uIgG4K顯示與抗體18.156.8近似的IC50，但無法完全阻斷PCSK9與LDLR的結合。 表13. 阻斷活性

4. 運用SPR技術偵測親和力 藉由使用實施例4第4.3.1節運用SPR技術偵測結合動力學常數中所述之同一方法測定，抗體12H11.1.uIgG4K的動力學親和力遠低於抗體18.156.8。結果示於表14。 表14.與人PCSK9結合的動力學親和力

5. LDL吸收實驗 根據實施例4第4.2節全人抗體的LDL吸收實驗中所述之方法，在HepG2細胞上對抗體18.156.8及12H11.1.uIgG4K進行LDL吸收活性的實驗(參見表15及圖31)。12H11.1.uIgG4K在HepG2細胞中顯示較低的恢復細胞LDL吸收的活性。 表15. LDL吸收實驗

雖然本公開案已具體示出且參考特定實施例(其中一些為較佳實施例)進行了描述，但熟習此項技術者應理解，如本申請所示可在不脫離本發明之精神及範圍內，可進行各種形式上及細節上的改變。The following description of the application is merely to illustrate various embodiments of the application. Therefore, the specific modifications discussed here should not be construed as limiting the scope of the application. Those skilled in the art can easily find many equivalents, changes and modifications without departing from the scope of the present application, and it should be understood that such equivalent embodiments are included in the scope of the present invention. All documents, including publications, patents and patent applications, cited in this application are hereby incorporated by reference in their entirety. Definitions The term "antibody" in the present invention includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody or bispecific (bivalent) antibody that binds to a specific antigen. A natural intact antibody contains two heavy chains and two light chains. Each heavy chain consists of a variable region and first, second, and third constant regions; each light chain consists of a variable region and a constant region. Mammalian heavy chains can be classified into α, δ, ε, γ, and μ, and mammalian light chains can be classified into λ or κ. Antibodies are in the shape of a "Y", with the neck of the "Y" structure consisting of the second and third constant domains of the two heavy chains, which are joined by disulfide bonds. Each arm of the "Y" structure includes the variable region and the first constant region of one of the heavy chains, which are combined with the variable and constant regions of a light chain. The variable regions of the light and heavy chains determine antigen binding. The variable region of each chain contains three hypervariable regions, called complementarity determining regions (CDRs) (the CDRs of the light chain (L) include LCDR1, LCDR2, and LCDR3, and the CDRs of the heavy chain (H) include HCDR1, HCDR2, and HCDR3) . The CDR boundaries of the antibodies and antigen-binding fragments disclosed in the present invention can be named or identified by Kabat, Chothia or Al-Lazikani nomenclature. (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J Mol Biol. Dec 5; 186( 3):651-63 (1985); Chothia, C. and Lesk, AM, J.Mol.Biol., 196,901 (1987); Chothia, C. et al., Nature. Dec 21-28;342(6252): 877-83 (1989); Kabat EA et al., National Institutes of Health, Bethesda, Md. (1991)). Of these, the three CDRs are separated by lateral contiguous portions called framework regions (FR), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding, but have multiple effector functions. Antibodies can be divided into several classes based on the amino acid sequence of the heavy chain constant region. Antibodies can be divided into five major classes or isoforms according to the presence or absence of α, δ, ε, γ, and μ heavy chains: IgA, IgD, IgE, IgG, and IgM, respectively. Several major antibody classes can also be divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain) or IgA2 (α2 heavy chain) and so on. The term "antigen-binding fragment" in this application refers to an antibody fragment formed from an antibody portion containing one or more CDRs or any other antibody fragment that binds to an antigen but does not have a complete antibody structure. Examples of antigen-binding fragments include, but are not limited to, such as diabodies, Fab, Fab', F(ab') ₂ , Fv fragments, disulfide bond-stabilized Fv fragments (dsFv), (dsFv) ₂ , Bispecific dsFv (dsFv-dsFv'), disulfide bond-stabilized bifunctional antibody (ds diabody), single-chain antibody molecule (scFv), scFv dimer (bivalent bifunctional antibody), bivalent single-chain antibody (BsFv), multispecific antibody, camelized single domain antibody (camelized single domain antibody), nanobody, domain antibody and bivalent domain antibody. An antigen-binding fragment can bind the same antigen as the parent antibody. In certain embodiments, an antigen-binding fragment may contain one or more CDRs from a particular human antibody grafted into framework regions from one or more different human antibodies. The "Fab" fragment of an antibody refers to that part of an antibody molecule that is composed of a light chain (including variable and constant regions) and a heavy chain's variable and constant regions through disulfide bonds. A "Fab'" fragment refers to a Fab fragment that includes part of the hinge region. "F(ab') ₂ " refers to a dimer of Fab. The "Fc" of an antibody refers to that part of the antibody that consists of the second and third constant regions of the heavy chain joined by disulfide bonds. The Fc portion of an antibody is responsible for a variety of effector functions such as ADCC and CDC, but does not participate in antigen binding. The "Fv" fragment of an antibody refers to the smallest fragment of an antibody that contains the complete antigen binding site. The Fv fragment consists of the variable region of one light chain and the variable region of one heavy chain. "Single-chain Fv antibody" or "scFv" refers to an engineered antibody in which the variable region of the light chain and the variable region of the heavy chain are linked directly or via a peptide chain (Huston JS et al., Proc Natl Acad Sci USA, 85 :5879 (1988)). "Single-chain antibody Fv-Fc" or "scFv-Fc" refers to an engineered antibody consisting of scFv linked to the Fc segment of an antibody. "Camelized single domain antibody (Camelized single domain antibody)", "heavy chain antibody" or "HCAb (Heavy-chain-only antibodies, HCAb)" all refer to antibodies containing two _VH domains without light chain ( Riechmann L. and Muyldermans S., J Immunol Methods. Dec. 10;231(1-2):25-38 (1999); Muyldermans S., J Biotechnol. Jun;74(4):277-302 ( 2001); WO94/04678; WO94/25591; US Patent No. 6,005,079). Heavy chain antibodies were originally derived from camelids (camels, dromedaries and llamas). Although lacking light chains, camelized antibodies have demonstrated fully functional antigen binding (Hamers-Casterman C. et al., Nature. Jun 3;363(6428):446-8 (1993); Nguyen VK. et al., "Heavy-chain antibodies in Camelidae; a case of evolutionary innovation," Immunogenetics. Apr;54(1):39-47 (2002); Nguyen VK. et al., Immunology. May;109(1) :93-101 (2003)). The variable regions (VHH domains) of heavy-chain antibodies are the smallest known antigen-binding units produced by adaptive immunity (Koch-Nolte F. et al., FASEB J. Nov;21(13):3490-8. Ev. June 15, 2007 (2007)). "Nanobody" refers to an antibody fragment consisting of a VHH domain from a heavy chain antibody and two constant regions CH2 and CH3. "Diabody" includes a small antibody fragment with two antigen-binding sites, wherein the fragment contains a _VH domain and a _VL domain ( _VH - _VL or _VH ) connected on the same polypeptide chain. -V _L ) (see, Holliger P. et al., Proc Natl Acad Sci US A. Jul 15;90(14):6444-8 (1993); EP404097; WO93/11161). The linker between the two domains is so short that the two domains on the same chain cannot pair with each other, thus forcing the two domains to pair with the complementary domains of another chain, forming two antibody binding sites. The two antibody combining sites can be targeted to bind the same or different antigens (or epitopes of antigens). "Domain antibody" refers to an antibody fragment that contains only one heavy chain variable region or one light chain variable region. In certain instances, two or more _VH domains are covalently joined by a polypeptide linker and form a bivalent domain antibody. The two _VH domains of bivalent domain antibodies can target the same or different antigens. In some embodiments, “(dsFv) ₂ ” contains three peptide chains: two _VH groups are connected by a polypeptide linker, and two _VL groups are connected by a disulfide bond. In certain embodiments, a "bispecific ds diabody" contains V _L1 -V _H2 (linked by a polypeptide linker) and V _H1 -V _L2 (also linked by a polypeptide linker), both at V _H1 and V _L1 are linked by a disulfide bond. "Bispecific dsFv" or "dsFv-dsFv" contains three polypeptide chains: V _H1 -V _H2 groups, in which the heavy chains of the two are connected by a polypeptide linker (such as: a long elastic linker), and are linked by two Sulfur bonds are combined with V _L1 and V _L2 groups respectively, and each pair of heavy and light chains paired by disulfide bonds has different antigen specificity. In certain embodiments, a "scFv dimer" is a bivalent diabody or a bivalent single chain antibody (BsFv) containing two _VH - _VL (linked by a polypeptide linker) groups that are dimerized , where the V _H of one group cooperates with the V _L of the other to form two binding sites that can target the same antigen (or antigenic determinant region of an antigen) or different antigens (or antigenic determinant regions) antigenic determinant region). In other embodiments, the "scFv dimer" is a bispecific bifunctional antibody comprising interconnected V _L1 -V _H2 (linked by a polypeptide linker) and V _H1 -V _L2 (linked by a polypeptide linker) , wherein V _H1 and V _L1 cooperate, V _H2 and V _L2 cooperate, and each cooperative pair has different antigen specificity. The term "fully human" used in this application, when used for an antibody or antigen-binding fragment, means that the antibody or antigen-binding fragment has or consists of a certain amino acid sequence, and the amino acid The acid sequences correspond to the amino acid sequences of antibodies produced by humans or human immune cells, or derived from non-human sources such as transgenic non-human animals using human antibody repertoires, or other sequences encoding human antibodies. In certain embodiments, fully human antibodies do not comprise amino acid residues (particularly antigen binding residues) derived from non-human antibodies. The term "humanized" as used in this application, when applied to antibodies or antigen-binding fragments, refers to the inclusion of CDRs derived from non-human animals, FR regions derived from humans, and constant regions (when applicable) derived from humans. ) antibody or antigen-binding fragment. Due to their reduced immunogenicity, humanized antibodies or antigen-binding fragments are useful as therapeutic agents in humans in certain embodiments. In some embodiments, the non-human animal is a mammal such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster. In some embodiments, the humanized antibody or antigen-binding fragment consists essentially entirely of human sequences, except that the CDR sequences are non-human. In some embodiments, the human-derived FR region may comprise the same amino acid sequence as the human antibody from which it was derived, or it may comprise some amino acid changes, e.g., no more than 10, 9, 8, 7 , 6, 5, 4, 3, 2 or 1 amino acid changes. In some embodiments, the amino acid change may be present only in the heavy chain FR region, only in the light chain FR region, or in both chains. In some preferred embodiments, the humanized antibody includes human FR1-3 and human JH and Jκ. The term "chimeric" as used in this application refers to an antibody or antigen-binding fragment having a portion of a heavy chain and/or light chain derived from one species and the rest of said heavy chain and/or light chain being derived from a different species . In one illustrative example, a chimeric antibody can include constant regions derived from a human and variable regions derived from a non-human animal, such as a mouse. As used herein, "PCSK9" refers to proprotein convertase subtilisin Kexin type 9, a native human proprotein convertase belonging to the proteinase K subfamily of the secretory subtilisin family. PCSK9 is synthesized as a lysozyme, undergoes autocatalytic intramolecular processing in the endoplasmic reticulum, and is thought to function as a proprotein convertase. PCSK9 plays a key role in regulating cholesterol levels in the blood. Gain-of-function (eg, S127R, F216L, and D374Y) mutations in PCSK9 may be associated with an autosomal dominant form of familial hypercholesterolemia in which PCSK9 mutations elevate LDL receptor levels (see eg, Burnett and Hooper, Clin Biochem Rev (2008) 29(1): 11-26, Benjannet et al. J. Biol. Chem., (2004) 279(47):48865-48875 and Fasano T et al., Atherosclerosis. (2009) 203(1):166 -71). The representative amino acid sequence of human PCSK9 is disclosed by GenBank accession number NP_777596.2, and the representative nucleic acid sequence encoding said human PCSK9 is disclosed by GenBank accession number FJ525880.1. In certain embodiments, the term PCSK9 includes PCSK9 disclosures of post-translational modifications of the PCSK9 amino acid sequence, such as glycosylation, pegylation of the PCSK9 sequence, splicing of the PCSK9 sequence from its signal sequence, or in the autocatalytic domain The PCSK9 sequence was cut out of its pro domain but not separated from the catalytic domain. As used herein, "LDL-C" refers to low-density lipoprotein cholesterol, and "HDL-C" refers to high-density lipoprotein cholesterol. LDL and HDL belong to five major lipoprotein groups: chylomicrons, very low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), low-density lipoprotein, and high-density lipoprotein (HDL) (in descending order Particles to the most dense (smallest particles). LDL ("bad" cholesterol containing particles) is able to transport lipid/sterol molecules such as cholesterol (i.e. LDL-C) to the arterial wall, attracting macrophages, thus inducing arterial Atherosclerosis. Conversely, HDL (the "good" cholesterol that contains particles) removes lipid molecules such as cholesterol (known as HDL-C) from macrophages on the arterial wall. Therefore, high levels of LDL-C Major risks of vascular disease (CVD) such as peripheral artery disease, coronary artery disease (CAD, such as angina, myocardial infarction (commonly known as heart disease), hyperlipidemia, hypercholesterolemia, hypertriglyceridemia), arterial Atherosclerosis, stroke, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, arrhythmia, congenital heart disease, valvular heart disease, myocarditis, aortic aneurysm, peripheral artery disease, obesity, liver and gallbladder disease, nephrotic syndrome, Hypothyroidism and venous thrombosis. "LDL-R" or "LDL receptor" as used in this application is a cell surface chimeric protein with 839 amino acids (with 21 amino acids removed from the signal peptide ), mediate the endocytosis of LDL-C and remove LDL-C from the blood. The representative amino acid sequence of human LDL-R is disclosed by GenBank Accession No. P01130.1, and it encodes human LDL-R The representative mRNA nucleotide sequence of is disclosed by GenBank Accession No. NM_000527.4. When PCSK9 is combined with the LDL receptor, the antibody is destroyed and cannot remove LDL-C from the blood. On the contrary, when PCSK9 is blocked, There will be more LDL receptors on the surface of the liver and more LDL cholesterol will be removed from the blood. "Anti-PCSK9 antibody" as used herein refers to an antibody capable of specifically binding to PCSK9 (such as human or monkey PCSK9), which Have sufficient affinity for diagnostic and/or therapeutic purposes. "Specific binding" or "specific binding" in this application refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and an antigen. In certain embodiments, the antibody or antigen-binding fragment thereof of the present application specifically binds to human and/or monkey PCSK9, and its binding affinity (K _D ) is ≤10 ^-6 M (eg: ≤5×10 ^-7 M , ≤2×10 ^-7 M, ≤10 ^-7 M, ≤5×10 ^-8 M, ≤2×10 ^-8 M, ≤10 ^-8 M, ≤5×10 ^-9 M, ≤2×10 ^{- 9} M, ≤10 ^-9 M, ≤10 ^-10 M). KD in this application refers to the ratio of dissociation velocity to association velocity (koff/kon), which can be determined by the method of surface surface plasmon resonance, for example Use an instrument such as Biacore. "Blocking binding" in this application " or the ability to "compete for the same epitope" refers to the ability of an antibody or antigen-binding fragment thereof to inhibit to any detectable extent the interaction of two molecules binding (eg, human PCSK9 and an anti-PCSK9 antibody). In certain embodiments, an antibody or antigen-binding fragment that blocks binding between two molecules inhibits the binding interaction between the two molecules by at least 50%. In certain embodiments, such inhibition may be greater than 60%, greater than 70%, greater than 80%, or greater than 90%. The "antigenic determinant region" used in this application refers to the amino acid or atomic group in the antigen molecule that binds to the antibody. If two antibodies exhibit competitive binding for the antigen, they are likely to bind to the same epitope on the antigen. For example, if the antibodies or antigen-binding fragments thereof provided herein block exemplified antibodies, such as WBP3011-2.6.6, 11.4, 18.156.8, 15.14.2, 17.72.3, 18.136.7, 19.3.8, 40409 and human PCSK9 binding, the antibody or antigen-binding fragment thereof can be regarded as binding to the same antigenic determinant region as those exemplified antibodies. The symbols in the antibody names used in this application have different representative meanings: "hIgG4" refers to an antibody with a constant region of the human IgG4 isotype; "uAb" refers to a human antibody, and uAb1, uAb2, etc. refer to the human antibody Different versions; "K" or "L" means that the antibody uses κ light chain or λ light chain. The "11.4" mentioned in this application refers to a fully human monoclonal antibody having a heavy chain variable region as shown in SEQ ID NO:73 and a light chain variable region as shown in SEQ ID NO:75. Antibody "11.4.1" is a hypopure line of 11.4. The "18.156.8" mentioned in this application refers to a fully human monoclonal antibody having a heavy chain variable region as shown in SEQ ID NO:77 and a light chain variable region as shown in SEQ ID NO:79. "18.156.8 (hIgG4)" is the 18.156.8 antibody with a constant region of the human IgG4 isotype. The "15.14.2" mentioned in this application refers to a fully human monoclonal antibody having a heavy chain variable region as shown in SEQ ID NO:81 and a light chain variable region as shown in SEQ ID NO:83. "15.14.2-uAb-IgG4L" is the 18.156.8 antibody with a human IgG4 isotype constant region. The "17.72.3" mentioned in this application refers to a fully human monoclonal antibody having a heavy chain variable region as shown in SEQ ID NO:85 and a light chain variable region as shown in SEQ ID NO:87. "17.72.3-uAb1-IgG4K" and "17.72.3-uAb2-IgG4K" are different versions of the 17.72.3 antibody with a constant region of the human IgG4 isotype. The "18.136.7" mentioned in this application refers to a fully human monoclonal antibody having a heavy chain variable region as shown in SEQ ID NO:89 and a light chain variable region as shown in SEQ ID NO:91. "18.136.7-IgG4K" is the 18.136.7 antibody with a human IgG4 isotype constant region. The "19.3.8" mentioned in this application refers to a fully human monoclonal antibody having a heavy chain variable region as shown in SEQ ID NO:93 and a light chain variable region as shown in SEQ ID NO:95. "19.3.8-IgG4L" and "19.3.8-uAb1-IgG4L" are 19.3.8 antibodies with human IgG4 isotype constant regions. The "40409" mentioned in this application refers to a fully human monoclonal antibody having a heavy chain variable region as shown in SEQ ID NO:97 and a light chain variable region as shown in SEQ ID NO:99. 40409 has improved affinity compared to its parental antibody 11.4. "40409 (hIgG4)" and "40409 (hIgG2)" are 40409 antibodies with human IgG4 isotype and IgG2 isotype constant regions, respectively. In this application, when "conservative substitution" is used for an amino acid sequence, it refers to replacing one amino acid residue with another amino acid residue with a side chain having similar physicochemical properties. For example, between hydrophobic side chain amino acid residues (such as Met, Ala, Val, Leu and Ile), between neutral hydrophilic side chain residues (such as Cys, Ser, Thr, Asn and Gln), acidic side chain Conservative substitutions were made between residues (eg Asp, Glu), between basic side chain amino acids (eg His, Lys and Arg) or between directional side chain residues (eg Trp, Tyr and Phe). It is known in this technology that conservative substitutions usually do not cause significant changes in the conformational structure of the protein, thus retaining the biological activity of the protein. When "percent sequence identity" is applied to an amino acid sequence (or nucleic acid sequence), it means that after alignment of the sequences and, if necessary, spacing is introduced to maximize the number of identical amino acids (or nucleic acids), the sequence in the candidate sequence In , the percentage of amino acid (or nucleic acid) residues identical to the reference sequence to the amino acid (or nucleic acid) residues of the candidate sequence. Conservative substitutions of such amino acid residues may or may not be considered to be the same residue. Tools that can be revealed by this technology, such as BLASTN, BLASTp (National Center for Biotechnology Information website (NCBI), see also, Altschul SF et al., J. Mol. Biol., 215:403-410 (1990) ; Stephen F. et al., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (European Bioinformatics Institute website, see, Higgins DG et al., Methods in Enzymology, 266:383-402 (1996 ); Larkin MA et al., Bioinformatics (Oxford, England), 23(21): 2947-8 (2007)) and ALIGN or Megalign (DNASTAR) software, the sequences are compared to determine the amino acid (or nucleic acid) sequence percent sequence identity. Those skilled in the art can use the default parameters of the tool or adjust the parameters appropriately according to the needs of the alignment, such as by selecting a suitable algorithm. "Effector function" as used in this application refers to the biological activity of the Fc region of an antibody in binding to its effectors such as the Cl complex and Fc receptors. Exemplary effector functions include complement-dependent cytotoxicity (CDC) induced by the interaction of the antibody with C1q on the C1 complex, antibody-dependent cell-mediated cytotoxicity induced by binding of the Fc region of the antibody to Fc receptors on effector cells (ADCC) and phagocytosis. "Treatment" or "therapy" of a condition includes preventing or alleviating a condition, reducing the rate of onset or development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition , to reduce or cease symptoms associated with a condition, to produce complete or partial reversal of a condition, to cure a condition, or a combination thereof. "Isolated" substances have been artificially altered from their natural state. When an "isolated" substance or component occurs in nature, it has been altered or separated from its original state, or both. For example, the polynucleotides or polypeptides naturally occurring in a living animal are not isolated, but if these polynucleotides or polypeptides are sufficiently separated from the substances that coexist in the natural state and exist in a sufficiently pure state, then Can be considered "separated". In certain embodiments, antibodies and antigen-binding fragments are at least 90%, 93%, 95%, 96%, 97%, 98%, 99% pure as determined by electrophoretic methods (e.g., SDS-PAGE, isoelectric focusing, , capillary electrophoresis), or chromatographic methods (such as ion-exchange chromatography or reversed-phase HPLC) to determine. The "vector" in the present invention refers to a delivery vehicle into which a polynucleotide encoding a certain protein can be operatively inserted and the protein can be expressed. Vectors can be used to transform, transduce or transfect host cells, so that the genetic material elements carried by them can be expressed in the host cells. Vectors include, for example: plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC), phage such as lambda phage or M13 phage , and animal viruses. Types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). The vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication. A vector may also include components to facilitate its entry into cells, including, but not limited to, viral particles, liposomes, or protein coats. The "host cell" in the present invention is a cell into which exogenous polynucleotides and/or vectors have been introduced. "PSCK9-mediated diseases or symptoms" in the present invention refers to diseases or symptoms caused or characterized by changes in PCSK9, such as changes in expression level and activity, and/or the presence of variants or mutations in PCSK9. Examples of diseases or conditions mediated by PCSK9 include, but are not limited to, dyslipidemia, hyperlipoproteinemia, hyperlipidemia; dyslipidemia; hypercholesterolemia, heart disease, stroke, coronary heart disease, atherosclerosis , peripheral vascular disease, claudication, type 2 diabetes, hypertension, cardiovascular disease or condition, inflammatory or autoimmune disease. Methods for identifying/diagnosing the aforementioned diseases or conditions are known in the art. For the application of the antibody or its antigen-binding fragment of the application in the treatment of CVD (such as acute myocardial infarction (AMI), acute coronary syndrome (ACS), stroke and cardiovascular death), the application uses "therapeutically effective dose" or "Effective dose" means the antibody or antigen binding thereof capable of lowering lipids (such as cholesterol) in plasma or serum, alleviating symptoms or markers associated with CVD conditions, preventing or delaying the development of CVD conditions, or a combination of the above The dose or concentration of the fragment. "Pharmaceutically acceptable" refers to a carrier, vehicle, diluent, excipient and/or salt that is generally chemically and/or physically compatible with the other ingredients in the formulation and physiologically compatible with the recipient compatible. Anti- PCSK9 antibody In certain embodiments, the present application provides exemplary fully human monoclonal antibodies 11.4, 18.156.8, 15.14.2, 17.72.3, 18.136.7, 19.3.8 and 40409, the CDR sequences thereof are shown in Table 1, and the heavy or light chain variable region sequences are also listed below. Table 1

11.4-VH: Amino acid sequence (SEQ ID NO: 73):

Nucleic acid sequence (SEQ ID NO: 74)

11.4-VL: Amino acid sequence (SEQ ID NO: 75):

Nucleic acid sequence (SEQ ID NO: 76)

18.156.8-VH amino acid sequence (SEQ ID NO: 77):

Nucleic acid sequence (SEQ ID NO: 78)

18.156.8-VL amino acid sequence (SEQ ID NO: 79):

Nucleic acid sequence (SEQ ID NO: 80)

15.14.2-VH amino acid sequence (SEQ ID NO: 81):

Nucleic acid sequence (SEQ ID NO: 82)

15.14.2-VL amino acid sequence (SEQ ID NO: 83):

Nucleic acid sequence (SEQ ID NO: 84)

17.72.3-VH amino acid sequence (SEQ ID NO: 85):

Nucleic acid sequence (SEQ ID NO: 86)

17.72.3-VL amino acid sequence (SEQ ID NO: 87):

Nucleic acid sequence (SEQ ID NO: 88)

18.136.7-VH amino acid sequence (SEQ ID NO: 89):

Nucleic acid sequence (SEQ ID NO: 90)

18.136.7-VL amino acid sequence (SEQ ID NO: 91):

Nucleic acid sequence (SEQ ID NO: 92)

19.3.8-VH amino acid sequence (SEQ ID NO: 93):

Nucleic acid sequence (SEQ ID NO: 94)

19.3.8-VL amino acid sequence (SEQ ID NO: 95):

Nucleic acid sequence (SEQ ID NO: 96)

40409-VH amino acid sequence (SEQ ID NO: 97):

Nucleic acid sequence (SEQ ID NO: 98)

40409-VL amino acid sequence (SEQ ID NO: 99):

Nucleic acid sequence (SEQ ID NO: 100)

In some embodiments, one or more of the CDR sequences described herein may be modified or altered such that the resulting antibody is improved in one or more properties relative to the original antibody (e.g., improved antigen binding, improved glycosylation pattern, reduced risk of glycosylation on CDR residues, increased pharmacokinetic half-life, pH sensitivity, and compatibility with conjugation), or comparable to the original antibody (i.e., except for the above modifications and Altered antibodies with the same CDR sequences), or at least substantially retain the antigen-binding properties of the original antibody. Those skilled in the art will appreciate that the CDR sequences provided in Table 1 may be modified to include one or more amino acid substitutions, thereby resulting in improved biological activity, eg, increased binding affinity to human PCSK9. For example, a library of antibody variants (such as Fab or FcFv variants) can be produced and displayed using phage display technology, followed by screening for antibodies with affinity to human PCSK9. In another example, computer software can be used to simulate the binding of the antibody to human PCSK9 and identify the amino acid residues on the antibody that form the binding interface. Substitution of such residues can be avoided to prevent loss of binding affinity, or can be targeted for substitution to result in stronger binding. In certain embodiments, at least one (or all) of the substitutions in the CDR sequences are conservative substitutions. In certain embodiments, the antibodies and antigen-binding fragments include one or more CDR sequences that are at least 80% (e.g., at least 85%, 88%, 90%, 91%) identical to the sequences listed in Table 1 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity, while retaining similar or even higher than its parental antibody and human PCSK9 Binding affinity, the parental antibody has substantially the same sequence, but its corresponding CDR sequence has 100% sequence identity with the sequences listed in Table 1. In certain embodiments, the anti-PCSK9 antibodies and antigen-binding fragments thereof are fully human. These fully human antibodies retain binding affinity to human PCSK9, preferably at levels similar to those of the exemplary antibodies: 11.4, 18.156.8, 15.14.2, 17.72.3, 18.136.7, 19.3.8 and 40409. The present application also includes antibodies and antigen-binding fragments thereof that compete for the same epitope as the anti-PCSK9 antibodies and antigen-binding fragments thereof of the present application. In certain embodiments, the antibody is present at less than 10 ⁻⁶ M, less than 10 ⁻⁷ M, less than 10 ^−7.5 M, less than 10 ⁻⁸ M, less than 10 ^−8.5 M, or less than 10 ^{− 9} M or lower than 10 ^-10 M IC ₅₀ value (i.e. half inhibitory concentration) block 11.4, 18.156.8, 15.14.2, 17.72.3, 18.136.7, 19.3.8 and 40409 with human or monkey PCSK9 combined. _IC50 values are determined by competitive assays such as ELISA and radioligand competition binding assays. In some embodiments, the anti-PCSK9 antibodies and antigen-binding fragments thereof described herein can be produced at no more than 10 ^-8 M, no more than 10 ^-9 M, or no more than 10 ^-10 M (e.g., ≤ 2.5×10 ^-8 M , ≤2×10 ^-8 M, ≤7.5×10 ^-9 M, ≤3.5×10 ^-9 M, ≤7×10 ^-10 M, ≤6×10 ^-10 M, ≤5×10 ^-10 M, ≤ 2.5×10 ^-10 M, ≤2×10 ^-10 M, ≤1.5×10 ^-10 M, ≤7.5×10 ^-11 M, ≤6.5×10 ^-11 M or ≤5.5×10 ^-11 M) binding affinity Specific binding (Kd) to human PCSK9 and/or monkey PCSK9 as measured by surface plasmon resonance binding or ELISA. Binding affinity can be expressed by _KD value, which is calculated by the ratio of off-rate to on-rate (koff/kon) when the binding of antigen and antigen-binding molecule reaches equilibrium. The antigen binding affinity (eg _KD ) may suitably be determined by suitable methods known in the art, including the use of instruments such as the surface plasmon resonance binding method such as Biacore (see for example Murphy, M. et al., Current protocols in protein science, Chapter 19, Unit 19.14, 2006). In certain embodiments, the antibodies and antigen-binding fragments thereof described herein bind to human PCSK9 at 0.01 nM-0.2 nM (such as 0.02 nM-0.2 nM, 0.02 nM-0.15 nM, 0.02 nM-0.05 nM, 0.01 nM-0.05 nM or 0.02nM-0.3nM) EC50 (ie half binding concentration) binding. The binding of the antibody to human PCSK9 can be determined by methods known in the art, such as sandwich methods, such as ELISA, Western blot or other binding assays. In an illustrative example, the antibody to be tested (i.e., the primary antibody) is bound to immobilized human PCSK9, followed by washing away unbound antibody and introducing a labeled secondary antibody, which is capable of binding to the primary antibody and thus capable of detecting the bound primary antibody. anti. The detection can be performed on a microplate reader plate when using immobilized PCSK9. In certain embodiments, the antibodies and antigen-binding fragments thereof described herein inhibit human PCSK9 with an IC of _0.5nM -3nM (eg, 0.5nM-2.5nM, 1nM-2.5nM, 1nM-2nM, or 1nM-1.5nM) Binding to its ligand, which was measured by a competition assay. In certain embodiments, the antibodies and antigen-binding fragments thereof bind monkey PCSK9 with a similar binding affinity as human PCSK9. For example, exemplary antibodies 11.4, 18.156.8, 15.14.2, 17.72.3, 18.136.7, 19.3.8, and 40409 bind monkey PCSK9 with similar affinities or _EC50 values as human PCSK9. In some embodiments, the anti-PCSK9 antibodies and antigen-binding fragments thereof also include immunoglobulin constant regions. In some embodiments, the immunoglobulin constant regions include heavy and/or light chain constant regions. The heavy chain constant region includes a CH1, CH1-CH2 or CH1-CH3 region. In some embodiments, the constant region may also include one or more modifications to obtain desired properties. For example, the constant region can be modified to reduce or deplete one or more effector functions, to enhance FcRn receptor binding, or to introduce one or more cysteine residues. In some embodiments, the anti-PCSK9 antibodies and antigen-binding fragments thereof have constant regions of the IgG4 isotype with reduced or depleted effector functions. Many tests are known for assessing ADCC or CDC activity, such as Fc receptor binding assay, complement C1q binding assay and cell lysis method, which can be easily selected by those skilled in the art. In some embodiments, the antibodies and antigen-binding fragments thereof are useful as base molecules for antibody-drug conjugates, bispecific or multivalent antibodies. The anti-PCSK9 antibody and its antigen-binding fragment described in this application can be monoclonal antibody, polyclonal antibody, fully human antibody, fully human antibody, humanized antibody, chimeric antibody, recombinant antibody, bispecific antibody , labeled antibody, bivalent antibody or anti-idiotypic antibody. Recombinant antibodies are antibodies produced in vitro using recombinant methods rather than animals. Bispecific antibodies, or diabodies, are artificial antibodies that have fragments of two different monoclonal antibodies that bind two different antigens. "Bivalent" antibodies and antigen-binding fragments thereof comprise two antigen-binding sites. The two antigen binding sites may bind the same antigen, or may each bind to a different antigen, in which case the antibody or antigen-binding fragment is "bispecific". In some embodiments, the anti-PCSK9 antibodies and antigen-binding fragments thereof described herein are fully human antibodies. In some embodiments, the fully human antibody is produced using recombinant methods. For example, transgenic animals, such as mice, can be prepared to carry transgenes or transchromosomes of human immunoglobulin genes and thus be capable of producing fully human antibodies following immunization with a suitable antigen, such as human PCSK9. Fully human antibodies can be isolated from such transgenic animals, or alternatively, can be produced by hybridoma technology, the spleen cells of the transgenic animals are fused with an immortal cell line to generate antibodies that secrete the fully human antibodies. Antibody-derived hybridoma cells. Exemplary transgenic animals include, but are not limited to, Omni rats in which expression of endogenous rat immunoglobulin genes has been inactivated and at the same time genetically engineered to contain functional recombinant human immunoglobulin loci ; Omni mice whose expression of endogenous mouse immunoglobulin genes has been inactivated and at the same time genetically engineered to contain recombinant human immunoglobulin loci with J-locus deletion and C-κ mutation. OmniFilc, which is a transgenic rat in which the expression of endogenous rat immunoglobulin genes has been inactivated and simultaneously genetically engineered to contain a single consensus, recombinant VkJk light chain and a functional heavy chain recombinant human immunoglobulin loci. For specific information, please refer to: Osborn M. et al., Journal of Immunology, 2013, 190: 1481-90; Ma B. et al., Journal of Immunological Methods 400-401 (2013) 78-86; Geurts A. et al., Science, 2009, 325:433; US Patent 8,907,157; European Patent 2152880B1; European Patent 2336329B1, all of which are incorporated herein by reference in their entirety. Other suitable transgenic animals can also be used, for example, HuMab mice (see in particular Lonberg, N. et al. Nature 368(6474): 856 859 (1994)), Xeno-mice (Mendez et al. Nat Genet., 1997, 15:146-156), TransChromo mice (Ishida et al. Cloning Stem Cells, 2002, 4:91-102) and VelocImmune mice (Murphy et al. Proc Natl Acad Sci USA, 2014, 111:5153-5158) , Kymouse transgenic mice (Lee et al. Nat Biotechnol, 2014, 32:356-363), and transgenic rabbits (Flisikowska et al. PLoS One, 2011, 6:e21045). In some embodiments, the anti-PCSK9 antibodies and antigen-binding fragments thereof described herein are camelized single chain domain antibodies, diabodies, scFv, scFv dimers, BsFv, dsFv, (dsFv)2, dsFv-dsFv', Fv fragment, Fab, Fab', F(ab')2, ds diabody, nanobody, domain antibody or bivalent domain antibody. In certain embodiments, the anti-PCSK9 antibodies and antigen-binding fragments thereof further comprise a conjugate. It is contemplated that the antibodies of the invention, or antigen-binding fragments thereof, may be linked to a variety of conjugates (see, e.g., "Conjugate Vaccines", Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr. (eds.), Carger Press, New York, (1989)). These conjugates can be linked to the antibody or antigen conjugate by other means such as covalent binding, affinity binding, intercalation, coordinate binding, complexation, binding, mixing or adding. In certain embodiments, the antibodies and antigen-binding fragments disclosed herein can be engineered to contain specific sites other than the binding portion of the epitope that can be used to bind one or more conjugates. For example, such sites may comprise one or more reactive amino acid residues, such as cysteine residues and histidine residues, to facilitate covalent attachment to the conjugate. In certain embodiments, the antibody may be linked to the conjugate indirectly, or via another conjugate. For example, the antibody or antigen-binding fragment thereof can bind biotin and then indirectly bind a second conjugate, which is linked to avidin. The conjugates can be detectable labels, pharmacokinetic modifying moieties, purification moieties or cytotoxic moieties. Examples of detectable labels may include fluorescent labels (such as luciferin, rhodamine, dandelion, phycoerythrin, or Texas Red), enzyme-substrate labels (such as horseradish peroxidase, alkaline phosphatase, luciferase, glucoamylase, lysozyme, sugar oxidase or β-D-galactosidase), radioactive isotopes (for example, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ³⁵ S, ³ H, ¹¹¹ In, ¹¹² In, ¹⁴ C, ⁶⁴ Cu, ⁶⁷ Cu, ⁸⁶ Y, ⁸⁸ Y, ⁹⁰ Y, ¹⁷⁷ Lu, ²¹¹ At, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁵³ Sm, ²¹² Bi, and ³² P , other lanthanides, luminescent labels), chromophore moieties, digoxin, biotin/avidin, DNA molecules or gold for detection. In certain embodiments, the conjugate may be a pharmacokinetic modifying moiety such as PEG, which helps prolong the half-life of the antibody. Other suitable polymers include, for example, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, ethylene glycol/propylene glycol copolymers, and the like. In certain embodiments, the conjugates can be purified moieties, such as magnetic beads. A "cytotoxic moiety" can be any agent that is harmful to cells or that may damage or kill cells. Exemplary cytotoxic moieties include, but are not limited to, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, Vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraxindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, Lucaine, tetracaine, lidocaine, propranolol, puromycin and its analogs, antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, arabinosin glycosides, 5-fluorouracil dacarba), alkylating agents (such as nitrogen mustard, thiotepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide , busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamminoplatinum (II) (DDP) cisplatin), anthracyclines (such as daunorubicin (formerly daunomycin) and doxorubicin), antibiotics such as dactinomycin (formerly known as actinomycin), bleomycin, mithramycin, and anthraninomycin (AMC)), and antimitotic agents ( eg vincristine and vinblastine). Polynucleotides and Recombinant Methods The application provides isolated polynucleotides encoding anti-PCSK9 antibodies and antigen-binding fragments thereof. In certain embodiments, the isolated polynucleotide comprises one or more nucleotide sequences as in Table 1, which encode the CDR sequences as in Table 1. In some embodiments, the isolated polynucleotide encodes a heavy chain variable region and comprises a sequence selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 34, and at least Homologous sequences with a sequence identity of 80% (eg, at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%). In some embodiments, the isolated polynucleotide encodes a light chain variable region and comprises a sequence selected from the group consisting of SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 36, and Homologous sequences of at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity . In certain embodiments, the percent identity is derived from the degeneracy of the genetic code, while the encoded protein sequence remains unchanged. Using recombinant techniques well known in the art, vectors comprising polynucleotides encoding said anti-PCSK9 antibodies and antigen-binding fragments thereof (for example comprising the sequences shown in Table 1) can be introduced into host cells for selection (amplification) DNA) or gene expression. In another embodiment, the antibody can be produced by homologous recombination methods known in the art. The DNA encoding the monoclonal antibody can be isolated and sequenced by known methods (for example, oligonucleotide probes can be used, which can specifically bind to the genes encoding the heavy and light chains of the antibody). Various carriers are available. Vector components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer sequence, a promoter (eg, SV40, CMV, EF-1α), and a transcription termination sequence . In some embodiments, the vector system includes mammalian, bacterial, yeast systems, etc., and will include plasmids such as, but not limited to, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, pACT2 and others can be obtained from the laboratory or the market carrier for sale. Suitable vectors may include plasmid or viral vectors (eg, replication defective retroviruses, adenoviruses, and adeno-associated viruses). Vectors comprising polynucleotides encoding the antibodies and antigen-binding fragments thereof can be introduced into host cells for cloning or gene expression. The host cells suitable for breeding or expressing the DNA in the vector in the present invention are prokaryotic cells, yeast or the above-mentioned higher eukaryotic cells. Prokaryotic cells suitable for use according to the invention include eubacteria such as Gram-negative or Gram-positive bacteria, for example, Enterobacteriaceae, such as Escherichia coli, Enterobacter, Erwinia, Klebsiella , Proteus, Salmonella, such as Salmonella typhimurium, Serratia, such as Serratia marcescens, and Shigella, and Bacillus, such as Bacillus subtilis and Bacillus licheniformis, Pseudomonas, such as Pseudomonas aeruginosa and Streptomyces. In addition to prokaryotic cells, eukaryotic microorganisms such as filamentous fungi or yeast can also be used as host cells for selection or expression of vectors encoding anti-PCSK9 antibodies. Saccharomyces cerevisiae, or baker's yeast, is the most commonly used lower eukaryotic host microorganism. However, many other genera, species and strains are commonly used and are suitable for use in the present invention, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as Kluyveromyces lactis, Kluyveromyces fragilis (ATCC 12,424) , Kluyveromyces bulgarica (ATCC 16,045), Kluyveromyces wilsonii (ATCC 24,178), Kluyveromyces (ATCC 56,500), Kluyveromyces drosophila (ATCC 36,906), Kluyveromyces thermotolerant and Maxk Ruvermyces; Yarrowia lipolytica (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesei (EP 244,234); Neurospora; Schwann's yeast; and filamentous fungi, such as Neurospora, Penicillium, Curvularia, and Aspergillus, such as Aspergillus and Aspergillus niger. Host cells suitable for expressing glycosylated antibodies or antigen-binding fragments thereof provided in the present invention are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. A variety of baculoviral strains and variants have been found, as well as corresponding permissive insect host cells, from hosts such as: Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito) , Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly) and silkworm. A variety of viral strains for transfection are publicly available, such as the Bm-5 variant of Autographa californica nuclear polyhedrosis virus and Bombyx mori nuclear polyhedrosis virus, which can be used in the present invention, especially Used to transfect Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts. However, of greatest interest are vertebral cells, and their culture (tissue culture) has become a well-known practice. Examples of useful mammalian host cells are, SV40 transformed monkey kidney cell line CV1 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 or 293 subcultured in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); Baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); Mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); Monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)) ; MRC 5 cells; FS4 cells; and human hepatoma cell line (Hep G2). In some preferred embodiments, the host cells are 293F cells. Host cells are transformed with the expression or cloning vectors described above that produce anti-PCSK9 antibodies and cultured in conventional nutrient media modified to induce promoters, select for transformed cells, or amplify the coding purpose sequence of genes. The host cells used in the present invention to produce the antibodies or antigen-binding fragments thereof can be cultured in various media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM, (Sigma)), RPMI-1640 (Sigma) and Dulbecco's Modified Eagle's Medium (DMEM), Sigma) can be used for culturing the host cells. Additionally, any of Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; /03430; WO 87/00195; or the medium described in US Patent Application Re. 30,985 can be used as the medium for the host cells. These media can be supplemented with necessary hormones and/or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium chloride, magnesium chloride and phosphate), buffers (such as HEPES), nucleotides (such as adenylic acid and thymine), antibiotics (such as gentamicin), trace elements (defined as inorganic compounds with final concentrations usually in the micromolar range), and glucose or its energy equivalent source. The medium may also contain any other necessary additives at appropriate concentrations known in the art. The conditions of the medium, such as temperature, pH and the like, are the conditions previously used to select the host cells for expression, and are well known to those skilled in the art. Using recombinant techniques, the antibodies can be produced intracellularly, in the periplasmic space, or secreted directly into the culture medium. If the antibody is produced intracellularly, particulate debris of host cells or lysed fragments are first removed, for example, by centrifugation or sonication. Carter et al., Bio/Technology 10:163-167 (1992) describe a method for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, the cell paste was dissolved in the presence of sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes or more. Cell debris was removed by centrifugation. If the antibody is secreted into the culture medium, the supernatant from the expression system is usually first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors such as PMSF to inhibit protein degradation, and antibiotics to prevent growth of incidental contaminants can be added at any of the preceding steps. Antibodies produced from the cells can be purified using purification methods such as hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography columns, ammonium sulfate precipitation, salting out, and affinity chromatography , where affinity chromatography is the preferred purification technique. The class of the antibody and the presence of any immunoglobulin Fc domains in the antibody determine the suitability of protein A as an affinity ligand. Protein A can be used to purify antibodies based on human γ1, γ2 or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is applicable to all murine isoforms as well as human γ3 (Guss et al., EMBO J. 5:1567 1575 (1986)). Agarose is the most commonly used matrix for affinity ligand attachment, but other matrices are also available. Mechanically stable matrices such as controlled-pore glass or poly(styrene)benzene allow faster flow rates and shorter processing times than with agarose. If the antibody contains a CH3 domain, it can be purified using Bakerbond ABX.TM resin (JT Baker, Phillipsburg, NJ). Other protein purification techniques can also be determined according to the antibodies obtained, such as fractionation in ion-exchange columns, ethanol precipitation, reverse-phase HPLC, silica gel chromatography, heparin-sepharose chromatography based on anion or cation exchange resins (such as polyaspartic acid column), chromatographic focusing, SDS-PAGE, and ammonium sulfate precipitation. After any preliminary purification steps, the mixture containing the antibody of interest and impurities can be treated by low pH hydrophobic interaction chromatography with a wash buffer at a pH of about 2.5-4.5, preferably at a low salt concentration (e.g., about 0 to 0.25M salt concentration). Kits The application provides kits comprising the anti-PCSK9 antibodies and antigen-binding fragments thereof. In some embodiments, the kit is used to detect the presence or level of PCSK9 in a biological sample. The biological sample may include serum. In some embodiments, the kit includes an anti-PCSK9 antibody and antigen-binding fragment thereof conjugated to a detectable label. In some embodiments, the kit includes unlabeled anti-PCSK9 antibodies and antigen-binding fragments thereof, and further includes a labeled secondary antibody capable of binding to the unlabeled anti-PCSK9 antibodies and antigen-binding fragments thereof. The kit may further include instructions for use and packaging separating the components in the kit. In some embodiments, the kits are used to treat, prevent or delay a disease or condition mediated by PCSK9. In some embodiments, the anti-PCSK9 antibodies and antigen-binding fragments thereof are linked to substrates or instruments for sandwich assays such as ELISA or immunochromatographic assays. Suitable substrates or instruments may be, for example, microwell plates and test strips. In some embodiments, the kit also includes one or more agents known to be beneficial in lowering cholesterol. Exemplary agents include statins, HMG-CoA reductase inhibitors other than statins, niacin (niacin), cholesterol absorption inhibitors, cholesteryl ester transfer protein (CETP), bile acid sequestrants, fibrates, Phytosterols; or regulators of lipid/lipid concentration ratios selected from small molecules, peptidomimetics, antisense RNA, small interfering RNA (siRNA), and natural or modified lipids. In certain embodiments, the cholesterol absorption inhibitor is ezetimibe or SCH-48461; CETP is evacetrapib, anacetrapib or dalcetrapib; the bile acid sequestrant is preferably colesevelam, cholestyramine or fibrates Ning is preferably fenofibrate, gemfibrozil, clofibrate, or bezafibrate; or a combination of the above agents. Pharmaceutical Compositions and Treatment Methods The present application further provides pharmaceutical compositions comprising the anti-PCSK9 antibodies and antigen-binding fragments thereof and one or more pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers used in the pharmaceutical compositions disclosed in this application may include, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous media, non-aqueous media, antimicrobial substances, etc. Osmotic substances, buffers, antioxidants, anesthetics, suspending/dispersing agents, chelating agents, diluents, adjuvants, adjuvants or non-toxic auxiliary substances, other components known in the art or multiple combinations of the above. Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants, emulsifiers or stabilizers, such as sugars and Cyclodextrin. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, thioglycolic acid, mercaptosorbitol, butylmethyl anise ether, butylated hydroxytoluene and/or propyl gallate. As disclosed herein, including one or more antioxidants such as methionine in a composition containing an antibody or antigen-binding fragment thereof disclosed herein will reduce oxidation of the antibody or antigen-binding fragment thereof. The reduction in oxidation prevents or reduces the loss of binding affinity, thereby improving antibody stability and extending shelf life. Accordingly, in certain embodiments, the compositions provided herein comprise one or more of the antibodies or antigen-binding fragments thereof and one or more antioxidants, such as methionine. The present invention further provides various methods, by mixing the antibody or antigen-binding fragment thereof provided in the present invention with one or more antioxidants, such as methionine, which can prevent the oxidation of the antibody or antigen-binding fragment thereof, prolong its shelf life and/or increase its activity. Further, pharmaceutically acceptable carriers may include, for example, aqueous media such as sodium chloride injection, Ringer's solution injection, isotonic glucose injection, sterile water injection, or glucose and lactated Ringer's solution. Injectable solutions, non-aqueous media such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antibacterial substances at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, Buffers such as: phosphate or citrate buffer, antioxidants such as: sodium bisulfate, local anesthetics such as: procaine hydrochloride, suspending and dispersing agents such as: sodium carboxymethylcellulose, hydroxypropyl methylcellulose or polyvinylpyrrolidone, emulsifiers such as polysorbate 80 (Tween-80), chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol bis(2- aminoethyl ether) tetraacetic acid), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid or lactic acid. Antimicrobial agents as carriers can be added to pharmaceutical compositions in multi-dose containers and include phenols or cresols, mercury agents, benzyl alcohol, chlorobutanol, methyl and propyl parabens, Thiamrosal, Methalkonium Chloride, and Ethonium Chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol or ethanol. Suitable nontoxic auxiliary substances may include, for example, emulsifiers, pH buffers, stabilizers, solubilizers, or substances such as sodium acetate, sorbitan laurate, triethanolamine oleate, or cyclodextrins . The pharmaceutical compositions may be liquid solutions, suspensions, emulsions, pills, capsules, lozenges, sustained release formulations or powders. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharine, cellulose, magnesium carbonate, and the like. In certain embodiments, the pharmaceutical compositions are prepared as injectable compositions. Injectable pharmaceutical compositions can be prepared in any conventional form, for example, liquid solvents, suspensions, emulsifications or solid forms suitable for liquid solvents, suspensions, or emulsifications. Injectable preparations may include ready-to-use sterile and/or pyrogen-free solutions, sterile dry solubles combined with solvents prior to use, such as lyophilized powders, including subcutaneous tablets, sterile suspensions ready for injection, mixed with Sterile dry insoluble products for vehicle combination, and sterile and/or pyrogen-free emulsions. The solvent can be aqueous or non-aqueous. In certain embodiments, the unit dose injection formulation is packaged in an ampoule, a tube or a syringe with a needle. As is known in the art, all preparations for parenteral administration should be sterile and pyrogen-free. In certain embodiments, sterile lyophilized powders can be prepared by dissolving an antibody or antigen-binding fragment thereof disclosed herein in an appropriate solvent. The solvent may contain an additional pharmacological component that increases the stability of the powder or reconstituted solution prepared from the powder, or improves the powder or reconstituted solution. Suitable excipients include, but are not limited to, water, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, dextrose, sucrose, or other suitable substances. The solvent may contain a buffer, such as citrate buffer, sodium or potassium phosphate buffer or other buffers known to those skilled in the art. In one embodiment, the pH of the buffer is neutral. The desired formulation is obtained by subsequent filter sterilization of the lyophilization under standard conditions well known in the art. In one embodiment, the resulting solvent is aliquoted into vials and lyophilized. Each vial can contain a single dose or multiple doses of the anti-PCSK9 antibody or antigen-binding fragment or composition thereof. The loading amount in each small tube can be slightly higher than that required for each dose or multiple doses (for example, 10% excess), so as to ensure accurate sampling and accurate administration. The lyophilized powder can be stored under appropriate conditions, such as at about 4°C to room temperature. The lyophilized powder is redissolved with water for injection to obtain a preparation for injection. In one embodiment, the lyophilized powder can be re-dissolved in sterile pyrogen-free water or other suitable liquid carriers. The precise amount is determined by the chosen therapy and can be determined empirically. Also provided are methods of treatment comprising administering to an individual in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment thereof described herein, thereby treating or preventing a condition or disorder associated with PCSK9. In another aspect, there is also provided a method of treating a condition in an individual that would benefit from an upregulated immune response comprising administering to said individual in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment thereof described herein. Therapeutically effective doses of the antibodies or antigen-binding fragments thereof provided herein depend on a variety of factors known in the art, such as body weight, age, past medical history, current therapy, the subject's health condition and potential for cross-infection, allergies, hypersensitivity and side effects, as well as the route of administration and the degree of tumor development. Dosages may be proportionally reduced or increased by those skilled in the art (eg, physician or veterinarian) according to these or other conditions or requirements. In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein can be administered at a therapeutically effective dose of about 0.01 mg/kg to about 100 mg/kg (eg, about 0.01 mg/kg, about 0.5 mg/kg kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg /kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, or about 100 mg/kg). In certain embodiments, the antibody or antigen-binding fragment thereof is administered at a dose of about 50 mg/kg or less, in certain embodiments, the dose is 10 mg/kg or less, 5 mg /kg or less, 3 mg/kg or less, 1 mg/kg or less, 0.5 mg/kg or less, or 0.1 mg/kg or less. A given dose may be given at multiple intervals, such as once a day, twice a day or more, twice a month or more, once a week, every two weeks, every three weeks, once a month, or every two weeks Once a month or more. In certain embodiments, the dose administered may vary over the course of the treatment. For example, in certain embodiments, the initial dose administered may be higher than the subsequent dose administered. In certain embodiments, the dose administered is adjusted during the course of treatment based on the response of the subject. Dosage regimens can be adjusted to achieve an optimal response (eg, a therapeutic response). For example, a single dose may be administered or multiple divided doses may be administered over time. The antibodies and antigen-binding fragments disclosed in the present invention can be administered by methods known in the art, such as injection (such as subcutaneous injection, intraperitoneal injection, intravenous injection, including intravenous drip, intramuscular injection or intradermal injection). Injection) or non-injection administration (eg, oral administration, nasal administration, sublingual administration, rectal administration or topical administration). Methods of Use The present application further provides methods of using the anti-PCSK9 antibodies or antigen-binding fragments thereof. In some embodiments, the present application provides methods of treating a PCSK9-mediated condition or disorder in an individual comprising administering a therapeutically effective amount of a PCSK9 antibody or antigen-binding fragment thereof described herein. In some embodiments, the individual is identified as having a disorder or condition that is likely to be responsive to a PCSK9 inhibitor. In certain embodiments, the individual is at risk of having or developing a disease or condition mediated by PCSK9 that exhibits one or more symptoms of the disease or condition, such as being overweight, Having elevated cholesterol levels, having an inherited mutation in the gene encoding LDL-R or APOB, or having a family history of these diseases or conditions. In certain embodiments, the individual is resistant or intolerant to another cholesterol-lowering agent (e.g., a statin) on treatment, and thus cholesterol levels cannot be effectively lowered to an acceptable level on that treatment level. In certain embodiments, the diseases or conditions mediated by PCSK9 include infectious diseases, such as severe cellulitis, gastroenteritis, sepsis, pneumonia, skin and soft tissue infections, pyelonephritis, viral infections, e.g., B Hepatitis, hepatitis C, viral infection with herpes virus, Epstein-Barr virus, HIV, cytomegalovirus, herpes simplex virus type 1, herpes simplex virus type 2, human papillomavirus, adenovirus, Kaposi's sarcoma associated Herpes virus epidemics, Torquetenovirus, JC virus or BK virus, or include inflammatory diseases such as Alzheimer's disease, ankylosing spondylitis, arthritis (osteoarthritis, rheumatoid arthritis (RA ), psoriatic arthritis), asthma, atherosclerosis, Crohn's disease, colitis, dermatitis, diverticulitis, fibromyalgia, hepatitis, irritable bowel syndrome (IBS), systemic lupus erythematosus (SLE), Nephritis, Parkinson's disease, and ulcerative colitis. The presence and level of LDL-C in the target biological tissue can indicate whether the individual from which the biological sample is derived is likely to respond to a PCSK9 inhibitor. The presence or level of LDL-C in the test biological sample from the individual can be determined using a variety of methods. Cholesterol levels are measured in milligrams (mg) per deciliter (dL) of blood in the United States, and millimoles (mmol) per liter (L) of blood in Canada and many European countries. In some embodiments, the presence or increased levels of LDL-C, total cholesterol, or non-HDL-C in said test biological sample indicates the likelihood of a response. As used herein, the term "up-regulation" refers to the total amount of cholesterol levels detected in a test sample using an antibody or antigen-binding fragment thereof described herein, compared to cholesterol levels in a reference sample detected using the same antibody. Increase by not less than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or more many. The reference sample can be a control sample obtained from a healthy or disease-free individual, or a healthy or disease-free sample obtained from the individual from which the sample to be tested is derived. The antibodies and antigen-binding fragments disclosed in the present invention can be administered alone or in combination with one or more other therapeutic means or substances. For example, the antibodies and antigen-binding fragments disclosed in the present invention can be combined with statins, HMG-CoA reductase inhibitors other than statins, nicotinic acid (nicotinic acid), cholesterol absorption inhibitors, cholesteryl ester transfer protein (CETP), bile Acid chelators, fibrates, phytosterols; or regulators of lipid/lipid concentration ratios selected from small molecules, peptidomimetics, antisense RNA, small interfering RNA (siRNA), and natural or modified lipids. In certain embodiments, the cholesterol absorption inhibitor is ezetimibe or SCH-48461; CETP is evacetrapib, anacetrapib or dalcetrapib; the bile acid sequestrant is preferably colesevelam, cholestyramine or fibrates Ning is preferably fenofibrate, gemfibrozil, clofibrate, or bezafibrate; or a combination of the above agents. In some of these embodiments, when the antibodies and antigen-binding fragments disclosed in the present invention are used in combination with one or more of the above-mentioned therapeutic substances, they can be administered simultaneously with the one or more of the above-mentioned therapeutic substances. In some of these embodiments, In the present invention, the antibody and the antigen-binding fragment can be administered simultaneously as part of the same pharmaceutical composition. However, antibodies and antigen conjugates "in combination" with other therapeutic substances need not be administered at the same time or in the same composition as the therapeutic substance. The meaning of "combined use" in the present invention also includes that antibodies and antigen conjugates administered before or after another therapeutic substance are also considered to be "combined with" the therapeutic substance, even if the antibody or antigen-binding fragment thereof is combined with the first therapeutic substance. The two substances are administered by different modes of administration. Where possible, other therapeutic substances used in combination with the antibodies disclosed in the present invention or their antigen-binding fragments can be administered according to the method in the product instructions of the other therapeutic substances, or refer to the surgeon's desk reference book 2003 (Physicians' Desk Reference , 57th Edition; Medical Economics Company; ISBN: 1563634457; 57th Edition (November 2002)), or other methods known in the art. The following examples are intended to better illustrate the present invention and should not be construed as limiting the scope of the present invention. All of the specific compositions, materials and methods described below, in whole or in part, are within the scope of the invention. Such specific compositions, materials and methods are not intended to limit the invention, but merely to illustrate that certain embodiments are within the scope of the invention. Those skilled in the art may develop equivalent compositions, materials and methods without adding inventive step and without departing from the scope of the present invention. It should be understood that various modifications made to the method of the present invention may still be included within the scope of the present invention. The inventors intend to include such variations within the scope of the present invention. Example 1 : Production of antigens and other proteins 1.1 Human and mouse PCSK9 The human and mouse PCSK9 genes were respectively assembled into the pcDNA3.3 vector, and the C-terminus was fused with a 6-His tag or a mouse Fc tag. These plasmids were respectively transfected into HEK293 cells using the transfection reagent PlasFect (Bioline USA, BIO-46026). Cell supernatants were collected after transfection. His-tagged protein was purified by Ni column (Qiagen Inc), and mouse Fc fusion protein was purified by Protein A column (MabSelect SuRe, GE). 1.2 Human LDL-R The LDL-R extracellular region gene was assembled into the pcDNA3.3 vector, and the C-terminus was fused with a 6-His tag. These plasmids were transfected into HEK293 cells using the transfection reagent PlasFect (Bioline USA, BIO-46026). Cell supernatant was collected after transfection, and LDL-R protein was purified with Ni column (Qiagen Inc) in the first step, and purified with ion exchange column in the second step. 1.3 Reference Antibody The sequence of the reference antibody BMK.115 is based on the 21B12 sequence in US Patent No. 8889834B2. Plasmids containing heavy and light chains were co-transfected into HEK293 cells. Cell supernatant was collected after transfection, and the antibody was purified by Protein A column (MabSelect SuRe, GE). Example 2 : Production of Antibodies 2.1 Immunization The transgenic rat OmniRat® (OMT) containing human immunoglobulin variable region genes was immunized to obtain fully human antibodies. Human PCSK9 protein plantar injection, once every three days. The first titer detection was performed after 6 immunizations, and immunization was performed once a week thereafter. 2.2 Serum titer detection The rat serum titer was detected by enzyme-linked immunosorbent assay (ELISA). Human PCSK9 protein was first diluted to 1 μg/ml with a coating solution with a pH value of 9.2, added to an ELISA plate (Nunc), and incubated overnight at 4°C. After the plate was washed and blocked, 200 μl of blocking solution 1×PBS/2% BSA was added to each well, and incubated at room temperature for 1 hour. Rat serum diluted 100 times was added to the initial well, then serially diluted with blocking solution at a ratio of 3 times, and incubated at room temperature for 1 hour. After washing the plate, the HRP enzyme-labeled secondary antibody mixture (Bethyl) of goat anti-rat IgG1 and goat anti-rat IgG2b was added, and incubated at room temperature for 1 hour. After washing the plate, add TMB substrate chromogenic solution, and then stop the color development with 2M hydrochloric acid. Absorbance at 450 nM was read using a microplate reader (Molecular Device). 2.3 Animal immunization and hybridoma generation Rats whose titer reached the requirement were selected, lymph nodes and spleens were removed, and transferred to a tissue grinder for grinding. B cells were counted after filtering through a 100-mesh sieve. Myeloma P3 cells were adjusted to an appropriate volume with medium and then counted. The two kinds of cells were resuspended and mixed according to the number of B cells:P3 = 1:1. The cell suspension was added to the fusion tank for electrofusion (BTX ECM2001). After fusion, the cells were transferred to medium containing 1/2 HA, and plated at a density of 5×10 ⁵ cells per plate. The titer of antigen-specific antibody in sera of immunized animals was detected by ELISA. Rats whose titer reached 312,500 were selected for fusion to generate hybridoma cells. 2.4 Hybridoma screening ELISA method to detect binding: First, dilute streptavidin to 1 μg/ml with a coating solution with a pH value of 9.2, add it to a 96-well microtiter plate (Nunc), and incubate overnight at 4°C . After sealing and washing the plate, biotin-labeled human PCSK9 protein was added at a concentration of 250 ng/ml, and incubated at room temperature for 1 hour. After washing the plate, add the supernatant of hybridoma cells and incubate at room temperature for 1 hour. After washing the plate, the HRP enzyme-labeled secondary antibody (Bethyl) mixture of goat anti-rat IgG1 and goat anti-rat IgG2b was added, and incubated at room temperature for 45 minutes. After washing the plate, add TMB substrate chromogenic solution, stop the color development with 2M hydrochloric acid, and then use a microplate reader (Molecular Device) to read the absorbance at 450 nm. Competitive ELISA: Add LDL-R to the microtiter plate (Nunc) and incubate overnight at 4°C. At the same time, the hybridoma cell supernatant was mixed with biotin-labeled human PCSK9 protein, the final concentration of PCSK9 protein was 250 ng/ml, and incubated overnight at 4°C. After the ELISA plate was washed and sealed, the mixture of hybridoma cell supernatant and PCSK9 protein was added, and incubated at room temperature for 1 hour. After washing the plate, HRP-labeled streptavidin was added. Finally, TMB substrate color development solution was added, and 2 M hydrochloric acid was used to stop the color development, and then the absorbance value at 450 nm was read using a microplate reader (Molecular Device). Antibody Screening In the first round of screening, hybridoma culture supernatants were used for antigen binding detection. A total of 13,000 hybridomas with specific binding to the antigen were screened out from the 4 fusions. These hybridoma cell lines were screened in further competition experiments. Through competition ELISA screening, 104 hybridoma cells were obtained, and the antibody secreted by them could block the combination of human PCSK9 and human LDL-R. These antigen-binding and blocking antibodies are purified from the supernatant of hybridoma cells. At the same time, these hybridoma cell lines were subselected. Subselections were detected using binding and competition ELISA and their subtypes were identified. The purified antibody was subjected to cell LDL uptake experiment. Binding and blocking activity will also be further detected with purified antibodies. Final candidate clones were selected based on binding affinity, ability to block PCSK9 binding to LDL-R, and ability to restore cellular LDL uptake. 2.5 Sub-selection of hybridoma cells The selected hybridoma cell lines were plated in 96-well plates at densities of 0.5, 1 and 5 cells per well. The individual wells were selected for detection by ELISA. For each hybridoma cell line, 3 subpure lines were preserved and frozen. 2.6 Detection of antibody subtypes ELISA was used to identify antibody isotypes (see Table 2). Goat anti-rat IgG1, IgG2a, IgG2b, IgG2c, and IgM antibodies (Bethyl) were diluted to 1 μg/ml with coating solution, respectively, added to 96-well microtiter plates (Nunc), and incubated overnight at 4°C. After the plate was washed and sealed, the hybridoma cell culture supernatant was added, and incubated at room temperature for 1 hour. After washing the plate, goat anti-human Kappa chain enzyme-labeled secondary antibody (Southern Biotech) or goat anti-human Lambda chain enzyme-labeled secondary antibody (Southern Biotech) were added, and incubated at room temperature for 45 minutes. After washing the plate, TMB substrate chromogenic solution was added, the color development was terminated with 2 M hydrochloric acid, and the absorbance at 450 nm was read with a microplate reader (Molecular Device). Table 2. Antibody subtypes

2.7 Antibody Purification The collected hybridoma supernatant was adjusted to pH 7.0, and then loaded onto a Protein A column (MabSelect SuRe, GE). Antibodies were eluted with Glycine and immediately neutralized with 1M Tris. The purified protein concentration was detected by Nano Drop (Thermal-Fisher). The purity of the protein was detected by SDS-PAGE (Invitrogen, NuPAGE 4%-12% Bis-Tris Gel) and HPLC-SEC (Agilent). Example 3 : Production of fully human antibodies 3.1 Hybridoma sequencing RNA was extracted from hybridoma cells using the Trizol kit (Invitrogen-15596018), and cDNA was amplified using the 5'-RACE kit (Takara-28001488), and then The cDNA was amplified by 3'-degenerate primers and 3'-linker primers (ExTaq: Takara-RR001B). The amplified fragment was inserted into pMD18-T vector (Takara-D101C) and sent for sequencing (Shanghai Boshang Company). The variable region sequences (amino acid sequence and nucleic acid sequence) of the selected antibodies 11.4, 18.156.8, 15.14.2, 17.72.3, 18.136.7, 19.3.8 and 40409 are shown in SEQ ID NO: 73-100 . 3.2 Expression and purification of fully human antibodies The variable region gene sequences of each antibody (human-mouse chimeric antibody) were cloned onto the pcDNA3.3 vector containing the human constant region genes. The heavy chain and light chain plasmids were transfected into HEK293 cells for antibody expression. Cell supernatant was collected by centrifugation. Antibodies were purified with Protein A (MabSelect SuRe, GE), and the purified antibodies were dialyzed into PBS. Antibody concentration by Nanodrop. The purity of the protein was detected by SDS-PAGE (Invitrogen, NuPAGE 4%-12% Bis-Tris Gel) and HPLC-SEC (Agilent). 3.3 Affinity maturation The saturation mutation library of 11.4 heavy chain CDR3 region was constructed and screened by ELISA method. Compared with the original pure line, the pure line whose PCSK9 affinity is improved by single point mutation is selected. The selected mutation sites were built into a combinatorial library. According to the results of ELISA binding experiment and SPR k _off sorting, the mutation combinations with improved affinity were selected. Using the 11.4 sequence as a template, two random mutation libraries were constructed. By performing two rounds of panning and screening on the library, mutations that can increase the affinity are selected and combined with the mutations obtained in the saturation mutation library, thereby further improving the affinity of the antibody. A saturation mutation library of 11.4 heavy chain CDR3 region was constructed and screened by ELISA method. Single point mutations selected pure lines with improved PCSK9 affinity compared to the original pure line (Figure 1). The selected mutation sites were built into a combinatorial library. According to the results of ELISA binding experiment and SPR k _off sorting, the mutation combinations with improved affinity were selected. The combination of mutations at the three sites in the CDR3 region can increase the affinity by 10 times, such as pure lines B4G2 and C1B4 (see Table 3). Table 3. Affinities of antibodies after affinity maturation

Using the 11.4 sequence as a template, two random mutation libraries were constructed. By performing two rounds of panning and screening on the library, 5 mutations capable of improving affinity were obtained. Three of the mutations were combined with the mutations in B4G2 to obtain three pure lines 40408, 40409 and 40410, thereby further increasing the affinity by 2 times. The affinity of the antibody 40409 after final affinity maturation was 2 times higher than that of the reference antibody BMK.115 (see Table 4). Table 4. Affinities of antibodies after affinity maturation

3.4 Transient expression of fully human antibody 3.4.1 18.156.8 (hIgG4) The fully human antibody 18.156.8-hIgG4) is characterized on SDS-PAGE under reducing conditions. The molecular weights are 25 kDa and 55 kDa, corresponding to the light chain of the antibody respectively and heavy chains. The main band on non-reducing SDS-PAGE corresponds to the complete IgG molecule with a molecular weight of about 150 KD. HPLC-SEC results showed that the purity of the antibody was 99.6% (Figure 3). The endotoxin content is less than 0.5 EU/mg. 3.4.2 40409 (hIgG4) Fully human antibody WBP301-40409 (hIgG4) was characterized on SDS-PAGE under reducing conditions with molecular weights of 25 kDa and 55 kDa, corresponding to the light chain and heavy chain of the antibody, respectively. The main band on non-reducing SDS-PAGE corresponds to the complete IgG molecule with a molecular weight of about 150 KD. HPLC-SEC results showed that the purity of the antibody was 98% (Figure 5). The endotoxin content is less than 0.5 EU/mg. 3.4.3 15.14.2-uAb-IgG4L The molecular weights of the fully human antibody 15.14.2-uAb-IgG4L on SDS-PAGE under reducing conditions are 25 kDa and 55 kDa, corresponding to the light chain and heavy chain of the antibody respectively. The main band on non-reducing SDS-PAGE corresponds to the complete IgG molecule with a molecular weight of about 150 KD. HPLC-SEC results showed that the purity of the antibody was 99.8% (Figure 7). The endotoxin content is less than 0.5 EU/mg. 3.4.4 17.72.3-uAb2-IgG4K The molecular weights of the fully human antibody 17.72.3-uAb2-IgG4K on SDS-PAGE under reducing conditions are 25 kDa and 55 kDa, corresponding to the light chain and heavy chain of the antibody respectively. The main band on non-reducing SDS-PAGE corresponds to the complete IgG molecule with a molecular weight of about 150 KD. HPLC-SEC results showed that the purity of the antibody was 98.9% (Figure 9). The endotoxin content is less than 0.5 EU/mg. 3.4.5 18.136.7-IgG4K The molecular weights of fully human antibody 18.136.7-IgG4K on SDS-PAGE under reducing conditions are 25 kDa and 55 kDa, corresponding to the light chain and heavy chain of the antibody respectively. The main band on non-reducing SDS-PAGE corresponds to the complete IgG molecule with a molecular weight of about 150 KD. HPLC-SEC results showed that the antibody purity was 99.2% (Figure 11). The endotoxin content is less than 0.5 EU/mg. 3.4.6 19.3.8-uAb1-IgG4L The molecular weights of fully human antibody 19.3.8-uAb1-IgG4L on SDS-PAGE under reducing conditions are 25 kDa and 55 kDa, corresponding to the light chain and heavy chain of the antibody respectively. The main band on non-reducing SDS-PAGE corresponds to the complete IgG molecule with a molecular weight of about 150 KD. HPLC-SEC results showed that the purity of the antibody was 99.9% (Figure 13). The endotoxin content is less than 0.5 EU/mg. Example 4 : Characterization of Candidate Antibodies 4.1 Binding and blocking activity of fully human antibody ELISA method to verify the binding activity of fully human antibody to PCSK9 and the activity of blocking PCSK9 binding to LDL-R are shown in Figures 14 and 15, respectively. See Table 5 and Table 6 for the results of the EC50 of the binding experiment and the IC50 of the blocking experiment. Candidate antibodies 18.156.8(hlgG4), 40409(hlgG4), 15.14.2-uAb-IgG4L and 17.72.3-uAb2-IgG4K showed binding and blocking activities comparable to reference antibodies BMK.115 and Repatha. Table 5. Antibody binding activity to PCSK9

Table 6. Blocking activity

4.2 LDL absorption experiment: HepG2 or Huh-7 cells were inoculated in 96-well plate at a density of 1×10 ⁵ per well in DMEM medium containing 10% FBS, and placed in a 37°C incubator. The next day, the DMEM culture medium containing 10% FBS was replaced with serum-free medium, and the wild-type PCSK9 or variant PCSK9 (D374Y) was mixed with a series of concentration gradient diluted antibodies and added to the corresponding wells, and incubated at 37°C for 1 Hour. The final concentrations of wild-type PCSK9 and variant PCSK9 (D374Y) were 20 μg/ml and 1.3 μg/ml, respectively. Bodipy fluorescently labeled LDL (Invitrogen L-3483) was added to the 96-well plate at a final concentration of 1.5 μg/ml. After incubating in a 37° C. incubator for 3 hours, the 96-well plate was taken out, the culture solution containing LDL was discarded, and cells were collected by trypsinization and washed twice. The fluorescence in the cells was detected by FACS, and the fluorescence intensity represented the absorption of LDL. The recovery rate of LDL absorption was calculated by the following formula: LDL absorption recovery rate (%) = (MFI _sample - MFI _LDL+Ag1H )/ (MFI _{LDL - MFILDL+Ag1H} ) × 100% . In the presence of wild-type and mutant PCSK9, the LDL uptake activity of antibodies 18.156.8 and 40409 was tested on HepG2 and Huh-7 cells (see Figures 16 and 17), and the IC50 values are shown in Table 7. Experiments have shown that antibodies 18.156.8 and 40409 can effectively restore the ability of HepG2 and Huh-7 cells to absorb LDL no matter in the presence of wild-type or mutant PCSK9. In the presence of wild-type PCSK9, the LDL uptake activity of antibodies 15.14.2, 17.72.3 and 19.3.8 was tested on HepG2 cells (Figure 18). The IC50 values are shown in Table 8. Experiments show that in the presence of wild-type PCSK9 Antibodies 15.14.2, 17.72.3 and 19.3.8 can effectively restore the LDL absorption ability of HepG2 cells under the condition of . Table 7. IC50 of Antibody Restoring LDL Uptake in HepG2 and Huh-7 Cells

Table 8. IC50 of Antibodies Restoring LDL Uptake in HepG2 Cells

4.3 Kinetic affinity 4.3.1 Detection of binding kinetic constants using SPR technology Biacore T200 (GE) was used to detect the binding affinity constants of antibodies to human PCSK9 and rhesus monkey PCSK9. Use Protein A or anti-IgG Fc antibody-conjugated chips to replenish the antibodies to be tested. Then different concentrations of human PCSK9 or rhesus monkey PCSK9 were injected onto the sensor chip, and the injection speed was 30 μl/min. The sample binding time was 180 s and the dissociation time was 1200 s. After each antigen binding, the chip was regenerated with ₂ M MgCl. The final binding-dissociation curve is the result after subtracting the signals of the reference channel Fc1 and the buffer channel. The experimental data were fitted with a 1:1 Langmiur model. A molecular weight of 85 KDa was used when calculating the molar concentration of PCSK9. 4.3.2 Binding reaction with rhesus monkey PCSK9 protein Dilute the mouse anti-His tag antibody (Genscript) to 1 μg/ml with the coating solution with a pH value of 9.2, add it to a 96-well microtiter plate, and incubate overnight at 4°C . After the plate was washed and blocked, rhesus monkey PCSK9-His (Sino Biological) protein was added at a concentration of 1 μg/ml and incubated at room temperature for 1 hour. After washing the plate, the antibody to be tested was added and incubated at room temperature for 1 hour. After washing the plate, the HRP enzyme-labeled secondary antibody (Bethyl) mixture of goat anti-rat IgG1 and goat anti-rat IgGb was added, and incubated at room temperature for 45 minutes. Finally, TMB substrate color development solution was added, and after the reaction, 2M hydrochloric acid was used to stop the color development, and then the absorbance value at 450 nm was read using a microplate reader (Molecular Device). The kinetic binding constants of the antibodies were detected by SPR experiments. The affinities of antibodies binding to human PCSK9 and monkey PCSK9 are shown in Table 9. Table 9. Kinetic constants of antibody binding to human PCSK9 and monkey PCSK9

4.4 Antibody Stability Detection in Serum The antibody to be tested was diluted with freshly isolated human serum (serum content > 95%), and incubated in a 37°C incubator for 0, 1, 3, 7, and 14 days respectively. At the end of each time period, after taking out the samples from the 37°C incubator, they were quickly frozen in an ethanol-dry ice bath and stored in a -80°C freezer. Samples were removed for rapid dissolution prior to stability testing. A 96-well ELISA plate was coated with streptavidin diluted in Na ₂ CO ₃ /NaHCO ₃ (pH 9.2) buffer and placed in a 4°C refrigerator. The next day, after the 96-well plate was washed with 0.1% PBST and blocked with 2% BSA/PBS for 1 hour, biotin-labeled PCSK9 was added. After incubation for 1 hour at room temperature and washing, a series of serially diluted samples were added. After incubation for 1 hour at room temperature and washing, HRP-labeled goat anti-human IgG antibody was added. After incubating at room temperature for 1 hour, wash, add TMB substrate, and stop with 2M HCl after color development. The absorbance at a wavelength of 450 nm was read with a microplate reader (Molecular Device). After the antibody was incubated in human serum at 37 degrees, its binding activity to PCSK9 was detected by ELISA (see Figure 19). Antibodies 18.156.8 and B4G2 were incubated in serum for 1 day, 3 days, 7 days and 14 days, and the binding activity was the same as that of unincubated antibodies, indicating that 18.156.8 and B4G2 could stably exist in human serum at 37 degrees for at least 14 days. sky. Antibodies 15.14.2, 17.72.3 and 19.3.8 had the same binding activity as unincubated antibodies after incubation in serum for 3 days. It shows that the antibody can stably exist in the serum for at least 3 days. Embodiment 5 : Animal experiment 5.1 Single injection drug effect in cynomolgus monkey Use LDLC and HDLC3 set (Roche) to detect low-density lipoprotein cholesterol LDL-C and high-density lipoprotein in monkey serum on Roche/Hitachi cobas c system Concentration of protein cholesterol HDL-C. Total cholesterol TCHO was detected with cholesterol FS kit (DiaSys). 5.1.1 Experiment 1: Six 3-4-year-old female cynomolgus monkeys, weighing between 2.6 and 2.9 kg, were randomly divided into 6 groups (one in each group). The six groups of monkeys received a single intravenous injection of 10 mg/kg or 30 mg/kg of BMK.115 or 18.156.8 (hIgG4) or 40409 (hIgG4) antibody. The day of administration was defined as the first day. The monkeys in each group were observed for 36 days after administration.

Note: In this experiment, "dose level" and "dose" are used interchangeably. ^a Unless otherwise stated, dosages represent the active ingredient. Efficacy of antibodies 18.156.8 and 40409 in lowering low-density cholesterol in cynomolgus monkeys. Antibodies BMK.115 and W301-18.156.8 can rapidly and durably reduce low-density lipoprotein cholesterol and total cholesterol levels in cynomolgus monkeys at doses of 10 mg/kg and 30 mg/kg, respectively. In the dose groups of 10 mg/kg and 30 mg/kg, compared with before injection, BMK.115 reduced LDL cholesterol to 83.4% and 89.6% respectively; 18.156.8 reduced LDL cholesterol to 79.5% and 83.5%. The maximum reduction was reached on day 8. Until the end of the detection, regardless of the 10 mg/kg or 30 mg/kg dose group, the low-density cholesterol of monkeys injected with 18.156.8 remained at a very low level; the control antibody BMK.115 was only in the 30 mg/kg dose group It can maintain the low-density lipoprotein level at a low level for 36 days, but in the 10 mg/kg dose group, the low-density lipoprotein level began to rise from the 24th day, and returned to the pre-administration level on the 28th day (Fig. 20A and 20B). Therefore, compared with the reference antibody BMK.115, 18.156.8 can maintain low-density lipoprotein at a lower level for a longer time. In the 10 mg/kg and 30 mg/kg dose groups, 40409 (hIgG4) reduced LDL cholesterol to 32.2% and 38.1%, respectively, compared to before injection (Fig. 20A and 20B). 18. In the monkeys administered with 156.8 (hIgG4) and 40409 (hIgG4) antibodies, no matter in the 10 mg/kg or 30 mg/kg dose group, the level of high-density lipoprotein cholesterol did not change significantly during the experiment. Among the monkeys administered with the reference antibody BMK.115, there was no significant change in the high-density cholesterol level of the monkeys in the 10 mg/kg dose group, but the high-density cholesterol level in the 30 mg/kg dose group was reduced by 30% compared with before administration (FIGS. 21A and 21B). 5.1.2 Experiment 2: 10 female cynomolgus monkeys aged 3-4, weighing between 2.5 and 3.5 kg, were randomly divided into 10 groups (one monkey in each group). Ten groups of monkeys received a single intravenous injection of 3 mg/kg or 10 mg/kg of Repatha or 15.14.2, 17.72.3, 18.136.7 or 19.3.8 antibodies. The day of administration was defined as the first day. The monkeys in each group were observed for 36 days after administration.

Note: In this experiment, "dose level" and "dose" are used interchangeably. ^a Unless otherwise stated, dosages represent the active ingredient. Antibodies 15.14.2, 17.72.3, 18.136.7, and 19.3.8 have low-density cholesterol-lowering efficacy in cynomolgus monkeys. Antibodies 15.14.2 and Repatha can rapidly and persistently reduce LDL cholesterol and total cholesterol levels after administration of 3 mg/kg and 10 mg/kg to cynomolgus monkeys respectively (Fig. 22A and 22B). Antibody 18.136.7 also significantly reduced LDL cholesterol (~50%) in the 3 mg/kg and 10 mg/kg dose groups. 17.72.3 and 19.3.8 reduced low-density cholesterol slightly in the 10 mg/kg dose group. In the dose groups of 3 mg/kg and 10 mg/kg, Repatha reduced low-density cholesterol to 80% and 77%, respectively, compared with before administration; 15.14.2 lowered low-density cholesterol in both dose groups to 77%. The maximum reduction is reached on days 8-16. In the 10 mg/kg dose group, monkeys injected with 15.14.2 maintained a very low level of LDL until the end of the test. On the other hand, the low-density lipoprotein level of monkeys administered with the control antibody Repatha began to rise from the 24th day, and returned to the pre-administration level on the 36th day (Fig. 22A and 22B). In the 3 mg/kg dose group, the low-density lipoprotein level of the monkeys administered Repatha began to rise to 80% on the 12th day, and had returned to the pre-administration level by the 20th day. 15.14.2 The level of low-density cholesterol in the monkeys administered with the drug remained below 50% before the drug until the 28th day. Therefore, compared with Repatha, in the two dose groups of 3 mg/kg and 10 mg/kg, 15.14.2 can maintain low-density lipoprotein at a lower level for a longer time. HDL cholesterol did not change significantly in all administered monkeys (FIGS. 23A and 23B). 5.2 Pharmacokinetics The drug exposure levels in vivo were obtained by measuring the concentrations of BMK.115, 18.156.8, 40409, 15.14.2, 17.72.3, 18.136.7, and 19.3.8 in animal serum. Blood samples were collected from all surviving cynomolgus monkeys at 0 hour (before dosing), and 0.5, 1, 2, 4, 24, 48, 96, 168, 336, 504, 672, 744 and 840 hours after dosing. About 2 mL of whole blood was collected from the cephalic vein or femoral vein of the animal, and placed in a blood collection tube without anticoagulant. After the blood sample was collected, it was allowed to stand at room temperature for at least 30 minutes. Serum was obtained by centrifugation at 2000 g for 10 minutes at about 4°C (completed within 2 hours after blood sample collection). Serum was transferred to labeled polypropylene sample tubes. Immediately freeze vertically in dry ice, and then store in a ≤-60° ultra-low temperature freezer. Serum samples were thawed rapidly prior to pharmacokinetic assays. After diluting the goat anti-human polyclonal antibody with Na ₂ CO ₃ /NaHCO ₃ buffer solution, coat the enzyme-labeled detection plate and incubate overnight at 4°C. After washing the plate with 0.1% Tween-PBS, add 2% BSA/PBS to block. The diluted cynomolgus monkey serum samples were added to the microtiter plate and incubated at room temperature for one hour. After washing the plate, add biotin-labeled goat anti-human IgG antibody and streptavidin-HRP to the ELISA plate successively, and incubate at room temperature for one hour respectively. Add TMB substrate, stop with 2M HCl after color development. Read the absorbance of each well at a wavelength of 450 nm. The concentration of antibody in serum was calculated by standard curve. Pharmacokinetic parameters include but are not limited to initial serum concentration (C ₀ ) and the area under the plasma concentration-time curve (AUC _0-840h ) from 0 hours to 840 hours after administration, using the validated WinNonlin program (PharsightVersion 6.2.1) for calculations. AUC _{0-840h was} calculated using the linear ascending/logarithmically descending trapezoidal rule of the non-compartmental method, limited to animals treated with the test article. In calculating the average value, BLQ (below quantitative value) was calculated as 0. The concentration of antibodies in the serum was detected by ELISA (Figures 24 and 25). See Table 10 for the C ₀ values and AUC _{0 - 840h} of BMK.115, 18.156.8(hIgG4), 40409(hIgG4) calculated from the antibody concentration in serum from 0 to 840 hours, and the antibody half-life. When the dose increased from 10 mg/kg to 30 mg/kg, the drug exposure levels (AUC _{0 - 840h} and/or C ₀ ) of 18.156.8 and 40409 increased proportionally, but the drug exposure level of BMK.115 increased with the dose The increase is not proportional. Table 10. Pharmacokinetic data I

See Table 11 for the C ₀ values and AUC _{0 - 840h} of Repatha, 15.14.2, 17.72.3, 18.136.7 and 19.3.8 calculated from the antibody concentration in serum from 0 to 840 hours, and the antibody half-life. In the two dose groups of 3 mg/kg and 10 mg/kg, the half-lives of antibodies 15.14.2, 17.72.3, 18.136.7 and 19.3.8 were longer than those of Repatha. When the dose increased from 10 mg/kg to 30 mg/kg, the drug exposure levels (AUC _{0 - 840h} and/or C ₀ ) of Repatha, 15.14.2, 17.72.3, 18.136.7 and 19.3.8 all increased proportionally . Table 11. Pharmacokinetic Information II

5.3 Immunogenicity Blood samples were collected from the cephalic vein or femoral vein of animals at 0 hour (before administration), and 336, 672, and 840 hours after administration. Antibodies 115, 18.156.8 and 40409 were diluted with Na ₂ CO ₃ /NaHCO ₃ buffer, plated (NUNC), and incubated overnight at 4°C. After washing the plate with 0.1% Tween-PBS, add 2% BSA/PBS to block. Monkey serum diluted in PBS was added to the microtiter plate and incubated at room temperature for 1 hour. After washing the plate, goat anti-cynomolgus IgG-HRP (no cross-reaction with human IgG) was added for incubation. After washing the plate, add TMB substrate, and stop with 2M HCl after color development. Read the absorbance of each well at a wavelength of 450 nm. The immunogenicity detection results of antibodies BMK.115, 18.156.8 (hIgG4) and 40409 (hIgG4) are shown in Figure 26. In monkey serum at 336, 672, and 840 hours after administration, the titers of anti-antibodies against BMK.115, 18.156.8 (hIgG4), and 40409 (hIgG4) were not significantly different from those before administration, indicating that a single injection of 10 BMK.115, 18.156.8 (hIgG4) or 40409 (hIgG4) antibodies elicited very low immunogenicity at mg/ml or 30 mg/ml doses. The immunogenicity detection results of antibodies Repatha, 15.14.2, 17.72.3, 18.136.7 and 19.3.8 are shown in Figure 27. In monkey serum at 336, 672, and 840 hours after administration, the titers of anti-antibodies against Repatha, 15.14.2, 17.72.3, 18.136.7, and 19.3.8 were not significantly different from those before administration, indicating that a single The immunogenicity of Repatha, 15.14.2, 17.72.3, 18.136.7 or 19.3.8 antibodies induced by injection at doses of 10 mg/ml or 30 mg/ml was very low. 5.4 Toxic death/dying: During the experiment, the health status of each animal was reported twice a day, once in the morning and once in the afternoon, except for animal inspection once when the animal arrived at the facility and on the day of autopsy. There were no unscheduled deaths of animals throughout the experiment. Detailed clinical observation: conduct one detailed clinical observation for all animals (including substitute animals) before the experiment, and conduct one observation for all experimental animals on the administration day (about 2 ± 0.5 hours after administration), and then conduct each observation during the experiment. Weekly detailed clinical observation. No drug-related clinical symptoms were observed throughout the experiment. Cage side observation: In the early stage of the experiment, from 2 days before the administration, all animals (including substitute animals) were observed once a day by the side of the cage. Once on the first day of the experiment, cage side observation was performed twice a day on the administration day (within 30 minutes after administration and about 6 ± 0.5 hours after administration), and cage side observation was performed once a day during the recovery period. Detailed clinical observations were scheduled during the same time period, and cage side observations were not performed. Body weight: Each animal was weighed once before the experiment. For experimental animals, body weight was weighed once before administration on the first day of the experiment, and weighed once a week during the subsequent experiment. No drug-related weight changes were observed, and all weight changes were within normal biological variation. Food intake: For all animals, the food intake of the animals was evaluated 2 days before the experimental administration and throughout the experimental administration and observation process. Daily food intake was assessed by monitoring the appetite of all animals (whether animals ate or not constituted assessment documentation). There were no drug-related changes in food intake. Example 6 : Activity comparison between antibody 18.156.8 and reference antibody 1. The method described in Example 1 and Example 2 was used to generate the reference antibody based on the sequences of 12H11.1 and 24B9.1 in the patent CN101932607 Reference antibodies 12H11.1.uIgG4K and 24B9.1.uIgG4L. The molecular weights of antibodies 12H11.1.uIgG4K and 24B9.1.uIgG4L on SDS-PAGE under reducing conditions were 25 kDa and 55 kDa, corresponding to the light chain and heavy chain of the antibody, respectively ( FIG. 28A ). The main band on non-reducing SDS-PAGE corresponds to the complete IgG molecule with a molecular weight of about 150 KD. HPLC-SEC results showed that the antibody purity of 24B9.1.uIgG4L was 96.8% ( FIG. 28B ). HPLC-SEC results showed that the antibody purity of 12H11.1.uIgG4K was 95.3% ( FIG. 28C ). 2. The binding to human PCSK9 determined by ELISA According to the method described in the hybridoma screening in Section 2.4 of Example 2, the ELISA method was used to verify the binding activity of reference antibodies 12H11.1.uIgG4K and 24B9.1.uIgG4L to human PCSK9 ( Figure 29). The combined EC50 values are shown in Table 12. Reference antibody 12H11.1.uIgG4K showed lower binding activity than antibodies 18.156.8 and BMK.115. Reference antibody 24B9.1.uIgG4L did not bind human PCSK9. Table 12. Binding activity

3. ELISA method to detect blocking According to the method described in the hybridoma screening in section 2.4 of Example 2, the ability of the reference antibodies 12H11.1.uIgG4K and 24B9.1.uIgG4L to block the combination of PCSK9 and LDLR was detected by competitive ELISA activity (Figure 30). See Table 13 for IC50 values. Antibody 24B9.1.uIgG4L showed no blocking activity. Antibody 12H11.1.uIgG4K showed similar IC50 to antibody 18.156.8, but could not completely block the binding of PCSK9 to LDLR. Table 13. Blocking activity

4. Using SPR technology to detect affinity By using the same method described in Example 4, section 4.3.1 using SPR technology to detect binding kinetic constants, the kinetic affinity of antibody 12H11.1.uIgG4K is much lower than that of antibody 18.156.8. The results are shown in Table 14. Table 14. Kinetic affinity for binding to human PCSK9

5. LDL absorption experiment According to the method described in the LDL absorption experiment of fully human antibody in Section 4.2 of Example 4, the LDL absorption activity experiment was carried out on HepG2 cells for antibodies 18.156.8 and 12H11.1.uIgG4K (see Table 15 and Figure 31). 12H11.1.uIgG4K showed lower activity in restoring cellular LDL uptake in HepG2 cells. Table 15. LDL Absorption Experiment

While the disclosure has been shown and described with reference to specific embodiments, some of which are preferred, it will be understood by those skilled in the art that the present disclosure may be made without departing from the spirit and scope of the invention as shown herein. Various changes in form and details can be made.

Figure 1 shows selected mutations in the 11.4 heavy chain CDR3 region. Figure 2 shows the SDS-PAGE staining results of the fully human antibody 18.156.8 (hIgG4) in the SDS-PAGE gel. M: protein molecular weight marker; Lane5: 18.156.8 (hIgG4), reducing condition; Lane6: 18.156.8 (hIgG4), non-reducing condition. Figure 3 shows the HPLC-SEC profile of the fully human antibody 18.156.8 (hIgG4), which shows a purity of 99.6%. Figure 4 shows the SDS-PAGE staining results of the fully human antibody 40409 (hIgG4) in the SDS-PAGE gel. M: protein molecular weight marker; Lane5: 40409 (hIgG4), reducing condition; Lane6: 40409 (hIgG4), non-reducing condition. Figure 5 shows the HPLC-SEC profile of the fully human antibody 40409 (hIgG4) showing a purity of 98%. Figure 6 shows the staining results of SDS-PAGE of fully human antibody 15.14.2-uAb-IgG4L in SDS-PAGE gel. M: protein molecular weight marker; Lane5: 15.14.2-uAb-IgG4L, reducing condition; Lane6: 15.14.2-uAb-IgG4L, non-reducing condition. Figure 7 shows the HPLC-SEC pattern of the fully human antibody 15.14.2-uAb-IgG4L showing a purity of 99.8%. Figure 8 shows the staining results of SDS-PAGE of fully human antibody 17.72.3-uAb2-IgG4K in SDS-PAGE gel. M: protein molecular weight marker; Lane5: 17.72.3-uAb2-IgG4K, reducing condition; Lane6: 17.72.3-uAb2-IgG4K, non-reducing condition. Figure 9 shows the HPLC-SEC pattern of the fully human antibody 17.72.3-uAb2-IgG4K showing a purity of 98.9%. Figure 10 shows the SDS-PAGE staining results of the fully human antibody 18.136.7-IgG4K in SDS-PAGE gel. M: protein molecular weight marker; Lane5: 18.136.7-IgG4K, reducing condition; Lane6: 18.136.7-IgG4K, non-reducing condition. Fig. 11 shows the HPLC-SEC profile of the fully human antibody 18.136.7-IgG4K showing a purity of 99.2%. Figure 12 shows the staining results of SDS-PAGE of fully human antibody 19.3.8-uAb1-IgG4L in SDS-PAGE gel. M: protein molecular weight marker; Lane5: 19.3.8-uAb1-IgG4L, reducing condition; Lane6: 19.3.8-uAb1-IgG4L, non-reducing condition. Figure 13 shows the HPLC-SEC profile of the fully human antibody 19.3.8-uAb1-IgG4L showing a purity of 99.9%. Figure 14 shows the ELISA experiment of the binding of fully human antibody to human PCSK9. Figure 15 shows the ELISA experiment of blocking the binding of PCSK9 and LDL-R by a fully human antibody. Figure 16 shows that fully human antibody 18.156.8 restores LDL in hepatocellular carcinoma (HepG2) cells and Huh-7 cells by binding to wild type (Figures 16A and 16B) and mutant PCSK9 (D374Y) (Figures 16C and 16D), respectively Absorb the experimental results. Figure 17 shows that fully human antibody 40409 restores LDL uptake experiments in hepatocellular carcinoma (HepG2) cells and Huh-7 cells by binding to wild-type (Figures 17A and 17B) and mutant PCSK9 (D374Y) (Figures 17C and 17D), respectively result. Figure 18 shows the experimental results of fully human anti-PCSK9 antibodies 15.14.2-uAb1-IgG4L, 17.72.3-uAb1-IgG4K and 19.3.8-uAb1-IgG4L in restoring LDL uptake in hepatocellular carcinoma (HepG2) cells. Figure 19 shows the stability of the fully human PCSK9 antibody (Figure 19A: 18.156.8; Figure 19B: B4G2; Figure 19C: 15.14.2, 19.3.8 and 17.72.3) indicated by the concentration determined by ELISA binding in serum sex. Figure 20 shows the percentage change of low-density lipoprotein (LDL-C) in cynomolgus monkeys after administration of antibodies 18, 156.8, 40409 and BMK.115. Figure 20A shows the results of a single injection of 10 mg/kg and Figure 20B shows the results of a single injection of 30 mg/kg. Figure 21 shows the percentage change of high-density lipoprotein (HDL-C) in cynomolgus monkeys after administration of antibodies 18, 156.8, 40409 and BMK.115. Figure 21A shows the results of a single injection of 10 mg/kg, and Figure 21 B shows the results of a single injection of 30 mg/kg. Figure 22 shows the percentage change of low-density lipoprotein (LDL-C) in cynomolgus monkeys after administration of antibodies 15.14.2, 19.3.8, 17.72.3, 18.136.7 and Repatha. Figure 22A shows the results of a single injection of 3 mg/kg and Figure 22B shows the results of a single injection of 10 mg/kg. Figure 23 shows the percentage change of high-density lipoprotein (HDL-C) in cynomolgus monkeys after administration of antibodies 15.14.2, 19.3.8, 17.72.3, 18.136.7 and Repatha. Figure 23A shows the results of a single injection of 3 mg/kg and Figure 23B shows the results of a single injection of 10 mg/kg. Figure 24 shows the concentration of antibody 18.156.8, 40409 or BMK.115 in cynomolgus monkey serum before and after administration determined by ELISA method. Figure 24A shows the results of a single injection of 10 mg/kg, and Figure 24B shows the results of a single injection of 30 mg/kg. Figure 25 shows the concentrations of antibodies 15.14.2 (hIgG4), 19.3.8 (hIgG4), 17.72.3 (hIgG4), 18.136.7 (hIgG4) or Repatha in cynomolgus monkey serum before and after administration determined by ELISA. Figure 25A shows the results of a single injection of 10 mg/kg and Figure 25B shows the results of a single injection of 30 mg/kg. Figure 26 shows anti-antibody (ADA) concentrations against antibodies 18.156.8 (hlgG4), 40409 (hlgG4) or BMK.115 in cynomolgus monkey serum before and after administration. Figures 26A, 26C and 26E show the results for a single injection of 10 mg/kg, and Figures 26B, 26D and 26F show the results for a single injection of 30 mg/kg. Figure 27 shows anti-antibody (ADA) concentrations against antibodies 15.14.2, 19.3.8, 17.72.3, 18.136.7 or Repatha in cynomolgus monkey serum before and after administration. Figures 27A, 27C, 27E, 27G and 27I show the results for a single injection of 3 mg/kg, and Figures 27B, 27D, 27F, 27H and 27J show the results for a single injection of 10 mg/kg. Figures 28A, 28B and 28C show the results of generation of reference antibodies 12H11.1 and 24B9.1. Figure 28A shows the SDS-PAGE results of 12H11.1.uIgG4K and 24B9.1.uIgG4L. M: protein molecular weight marker; Lane1: 24B9.1.uIgG4L, reducing condition; Lane2: 12H11.1.uIgG4K, reducing condition; Lane3: 24B9.1.uIgG4L, non-reducing condition; Lane4: 12H11.1.uIgG4K, non-reducing condition condition. Figures 28B and 28C show HPLC-SEC detection of 24B9.1.uIgG4L and 12H11.1.uIgG4K. Figure 29 shows the comparison of the binding of antibody 18.156.8 and reference antibodies 24B9.1, 12H11.1 and BMK.115 to human PCSK9 determined by ELISA method. Fig. 30 shows the comparison results of antibody 18.156.8 and reference antibodies 24B9.1, 12H11.1 and BMK.115 blocking the binding of PCSK9 and LDLR determined by ELISA method. Figure 31 shows the ability of antibody 18.156.8 and reference antibodies 12H11.1 and BMK.115 to restore LDL uptake in HepG2 cells.

<110> Shanghai Wuming Biotechnology Co., Ltd. (WUXI BIOLOGICS (SHANGHAI) CO.,LTD.)

<120> Novel anti-PCSK9 antibodies

<130> 053674-8009TW01

<140> TW 106131295

<141> 2017-09-12

<150> CN 201610836152.4

<151> 2016-09-20

<160> 100

<170> PatentIn Version 3.5

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Claims (18)

An isolated antibody or antigen-binding fragment thereof, comprising: a heavy chain variable region comprising HCDR1 as shown in SEQ ID NO: 13, HCDR2 as shown in SEQ ID NO: 15, and HCDR2 as shown in SEQ ID NO: 17 and the light chain variable region comprising LCDR1 as shown in SEQ ID NO: 19, LCDR2 as shown in SEQ ID NO: 21 and LCDR3 as shown in SEQ ID NO: 23; and wherein the separation The antibody or antigen-binding fragment thereof specifically binds to proprotein convertase subtilisin Kexin type 9 (PCSK9). The antibody or antigen-binding fragment thereof according to claim 1, comprising: a heavy chain variable region as shown in SEQ ID NO:77; and a light chain variable region as shown in SEQ ID NO:79. The antibody or antigen-binding fragment thereof as claimed in claim 1, which is a fully human monoclonal antibody. The antibody or its antigen-binding fragment according to claim 1, which is a diabody, scFv, scFv dimer, BsFv, dsFv, (dsFv)2, dsFv-dsFv', Fv fragment, Fab, Fab', F(ab')2, ds diabody, domain antibody or bivalent domain antibody. The antibody or antigen-binding fragment thereof according to claim 1, further comprising an immunoglobulin constant region. The antibody or antigen-binding fragment thereof according to claim 1, further comprising a conjugate. An isolated polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6. A vector comprising the isolated polynucleotide according to claim 7. A host cell comprising the vector according to claim 8. A method for expressing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, comprising culturing the host cell according to claim 9 under the condition of expressing the isolated polynucleotide according to claim 7. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6. A use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, which is used to prepare a medicine for blocking the combination of PCSK9 and LDL-R in an individual. The use according to claim 12, wherein the individual is identified as suffering from a disorder or condition that is likely to respond to a PCSK9 inhibitor. Such as the use of claim 13, wherein the individual is identified as being tested in the organism from the individual Serum LDL cholesterol, total cholesterol and/or non-HDL cholesterol levels in the sample are upregulated. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 and one or more pharmaceutically acceptable carriers. The use according to claim 12, wherein the individual has elevated serum LDL cholesterol, total cholesterol and/or non-HDL cholesterol levels. Such as the use of claim 12, wherein the medicine is used to treat cardiovascular diseases, inflammatory diseases and infectious diseases. As the use of claim 17, wherein the infectious disease is sepsis.

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